MX2015004560A - Pyrrolobenzodiazepines and conjugates thereof. - Google Patents

Pyrrolobenzodiazepines and conjugates thereof.

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MX2015004560A
MX2015004560A MX2015004560A MX2015004560A MX2015004560A MX 2015004560 A MX2015004560 A MX 2015004560A MX 2015004560 A MX2015004560 A MX 2015004560A MX 2015004560 A MX2015004560 A MX 2015004560A MX 2015004560 A MX2015004560 A MX 2015004560A
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Philip Wilson Howard
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Spirogen Sàrl
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
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Abstract

A compound which is selected from A, B and C, and salts and solvates thereof, as well as conjugates thereof with cell binding agents.

Description

PIRROLOBENZODIAZEPINAS AND CONJUGATES OF THE SAME FIELD OF THE INVENTION The present invention relates to pyrrolobenzodiazepines (PBDs), in particular, to pyrrolobenzodiazepines having a labile C2 protecting group, in the form of a linker to a cell binding agent.
BACKGROUND OF THE INVENTION Pyrrolobenzodiazepines Some pyrrolobenzodiazepines (PBDs) have the ability to recognize and bind to specific DNA sequences; the preferred sequence is PuGPu. The first PBT antitumor antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al. , J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and more than 10 synthetic routes have been developed for a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)).; Antonow, D. and Thurston, D.E., Chem. Rev. 2011 111 (4), 2815-2864). Members of the family include abeimycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), Chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206. (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et. al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazetramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neotramycin A and B (Takeuchi, et al., J. Antibiotics, 29 , 93-96 (1976)), porotramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), protracarcin (Shimizu, et al., J. Antibiotics, 29, 2492-2503 (1982 ); Langlcy and Thurston, J.
Org. Chem., 52, 91-97 (1987)), sibanomycin (DC-102) (Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41 , 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). The PBDs are of the general structure: They differ in the number, type and position of the substituents, both in their aromatic A rings and in their C rings of pyrrolo, and in the degree of saturation of the C ring. In the B ring there exists either an imine (N = C), a carbinolamine (NH-CH (OH)), or a methyl ether of carbinolamine (NH-CH (OMe)) at position N10-Cll which is the electrophile center responsible for the DNA alkylation. All known natural products have a configuration (S) in the chiral Clla position that gives them a rightward turn when viewed from ring C to ring A. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of the B-shaped DNA, leading to a tight fit in the binding site (Kohn, in Antibiotics III, Springer-Verlag, New York, pp 3-11 (1975), Hurlcy and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove allows them to interfere with DNA processing, hence their use as antitumor agents.
A particularly advantageous pyrrolobenzodiazepine compound is described by Gregson et al. . { Chem. Commun. 1999, 797-798) as the compound 1, and by Gregson et al. (J. Med. Chem. 2001, 44, 1161-1174) as the compound 4a. This compound, also known as SG2000, is shown below: WO 2007/085930 describes the preparation of dimeric PBD compounds having linkage groups for connection to a binding agent to cell, such as an antibody. The linker is present in the bridge that links the monomeric PBD units of the dimer.
The present inventors have described dimeric PBD compounds that have linking groups for connection to a cell binding agent, such as an antibody, in WO 2011/130613 and WO 2011/130616. The linker in these compounds is linked to the PBD core via the C2 position, and are generally cleaved by the action of an enzyme on the linking group.
Antibody-drug conjugates Antibody therapy has been established for the targeted treatment of patients with cancer, immunological and angiogenic disorders (Cárter, P. (2006) Nature Reviews Immunology 6: 343-357). The use of antibody-drug conjugates (ADCs), ie, immunoconjugates, for the local administration of cytotoxic or cytostatic agents, ie, drugs to destroy or inhibit tumor cells in the treatment of cancer, is directed to the administration of the drug portion to tumors, and intracellular accumulation therein, while systemic administration of these unconjugated drug agents can result in unacceptable levels of toxicity to normal cells (Xie et al (2006) Expert Opin. Biol. Ther. 6 (3): 281-291; Kovtun et al (2006) Cancer Res. 66 (6): 3214-3121; Law et al (2006) Cancer Res. 66 (4): 2328-2337; Wu et al (2005) Nature Biotech. 23 (9): 1137-1145; Lambert J. (2005) Current Opin. in Pharmacol. 5: 543-549; Hamann P. (2005) Expert Opin. Ther. Patents 15 (9): 1087-1103; Payne, G. (2003) Cancer Cell 3: 207-212; Trail et al (2003) Cancer Immunol. Immunother. 52: 328-337; Syrigos and Epenetos (1999) Anticancer Research 19: 605-614).
Thus, maximum efficiency is sought with minimum toxicity. Efforts to design and refine ADCs have been based on the selectivity of monoclonal antibodies (mAbs) as well as on the mechanism of drug action, drug binding, drug / antibody ratio (loading) and drug release properties. (Junutula, et al., 2008b Nature Biotech., 26 (8): 925-932; Dom et al. (2009) Blood 114 (13): 2721-2729; US 7521541; US 7723485; W02009 / 052249; McDonagh ( 2006) Protein Eng. Design &Sel.19 (7): 299-307; Doronina et al (2006) Bioconj Chem. 17: 114-124; Erickson et al (2006) Cancer Res. 66 (8): 1 -8; Sanderson et al (2005) Clin Cancer Res 11: 843-852; Jeffrcy et al (2005) J. Med. Chem. 48: 1344-1358; Hamblett et al (2004) Clin Cancer Res. : 7063-7070). The drug portions can confer their cytotoxic and cytostatic effects by mechanisms that include binding to tubulin, DNA binding, inhibition of proteasome and / or topoisomerase. Some cytotoxic drugs tend to be inactive or less active when conjugated with large antibodies or protein receptor ligands.
The present inventors have developed particular PBD dimers with linking groups for the formation of PBD conjugates with cell binding agents, and in particular antibody and PBD conjugates.
BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides a compound, which is selected from A: TO B: and C: and salts and solvates thereof.
WO 2011/130615 describes the compound 26: which is the compound of origin of A. Compound A comprises this PBD with a linker for connection to a cell-binding agent. The cell binding agent provides a series of portions of ethylene glycol to provide solubility, which is useful in the synthesis of conjugates.
WO 2010/043380 and WO 2011/130613 describe compound 30: WO 2011/130613 also describes compound 51: Compound B differs from compound 30 by having only one (CH2) 3 chain between the PBD portions, instead of a (CH2) 5 chain, which reduces the lipophilicity of the released PBD dimer. The linking group binds to the C2-phenyl group in the para position instead of the meta position.
WO 2011/130613 describes the compound 93: Compound C differs from it in two aspects. The cell-binding agent provides a greater number of portions of ethylene glycol to provide solubility, which is useful in the synthesis of conjugates, and the phenyl substituent provides two instead of a carbon atom. oxygen, which also helps solubility. The structure of compound C can also mean that it binds more strongly in the minor groove.
Compounds A, B and C have two sp2 centers in each C ring, which may allow a stronger bond in the minor groove of DNA, than for compounds with only one sp2 center in each C ring.
A second aspect of the present invention provides a conjugate of formula ConjA: O Conj C: ConjC wherein CBA represents a cell binding agent. The binding to the portion shown is by means of a free S (active thiol) in the cell binding agent.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the result of the cytotoxicity of ADC2A that was evaluated in a test thereof and is explained in Example 10 below.
Figure 2 shows the effect on mean tumor volume in groups of 10 mice dosed with ADC1A at 0.3 (yellow) or 1.0 mg / kg (purple) compared to vehicle controls (black) or naked antibody (blue).
Figure 3 shows the effect on mean tumor volume in groups of 10 mice dosed with ADClB at 0.3 (gray) or 1.0 mg / kg (purple) compared to vehicle controls (black) or naked Ig (blue).
DETAILED DESCRIPTION OF THE INVENTION The present invention provides a PBD dimer with a linker connected through the C2 position in one of the PBD portions suitable for the formation of a PBD dimer conjugated via the linker to a cell binding agent.
The present invention is suitable for use in providing a PBD compound to a preferred site in a subject. The conjugate allows the release of an active PBD compound that does not retain any part of the linker. There is no present tip that could affect the reactivity of the PBD compound. In this way, the ConjA would release the RelA compound: RelA The ConjB would release the compound RelB: And the ConjC would release the RelC compound RelC A further aspect of the present invention is the compound RelB, and salts and solvates thereof.
A further aspect of the present invention is the RelC compound, and salts and solvates thereof.
The specified link between the PBD dimer and the cell-binding agent, eg, antibody, in the present invention is preferably extracellularly stable. Prior to transport or administration in a cell, the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e., the antibody remains bound to the drug portion. The linkers are stable outside the target cell and can be excised at a certain effective rate inside the cell. An effective linker: (i) will maintain the specific binding properties of the antibody; (ii) will allow intracellular administration of the conjugate or drug portion; (iii) it will remain stable and intact, that is, it will not be split, until the conjugate has been administered or transported to its chosen site as a target; and (iv) maintain a cytotoxic, destructive effect of cells or a cytostatic effect of the drug portion of PBD. The stability of the ADC can be measured by conventional analytical techniques such as mass spectroscopy, HPLC, and the separation technique / LC / MS analysis.
The administration of the compounds of formulas RelA, RelB or RelC is achieved at the desired activation site of conjugates of formulas ConjA, ConjB or ConjC through the action of an enzyme, such as cathepsin, in the linking group, and in particular in the dipeptide portion of valine-alanine.
Cell binding agent A cell-binding agent can be of any type, and include peptides and not peptides. These may include antibodies or a fragment of an antibody that contains at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient transport molecules, or any other cell or substance binding molecule. .
Peptides In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20, contiguous amino acid residues. In this embodiment, it is preferred that a cell binding agent be linked to a pyrrolobenzodiazepine compound monomeric or dimeric.
In one embodiment, the cell-binding agent comprises a peptide that binds to integrin anb6. The peptide may be selective for anb6 with respect to XYS.
In one embodiment, the cell-binding agent comprises the A20FMDV-Cys polypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, a variant of the A20FMDV-Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are substituted with another amino acid residue. Additionally, the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.
Antibodies The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity (Miller et al (2003) Jour. Of Immunology 170: 4854-4861). The antibodies can be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs in multiple antibodies. Each antibody that binds specifically to a different epitope has a different structure. In this way, an antigen can have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, ie, a molecule that contains an antigen binding site that immunospecifically binds to an antigen of a target of interest or part thereof, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (eg, IgG, IgE, IgM, IgD and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Immunoglobulins can be derived from any species, which includes human, murine or rabbit origin.
"Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding region or variable region thereof. Examples of antibody fragments include fragments Fab, Fab ', F (ab') 2 and scFv; diabodies; linear antibodies; fragments produced by a library of Fab expression, anti-idiotypic (anti-Id), CDR (complementarity determining region) and epitope-binding fragments of any of the foregoing which immunospecifically bind to antigens of cancer cells, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
The term "monoclonal antibody", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. which may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Additionally, unlike polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to its specificity, monoclonal antibodies they are advantageous because they can be synthesized without being contaminated by other antibodies. The adjective "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies that will be used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al (1975) Nature 256: 495, or can be prepared by recombinant DNA methods (cf. US 4816567). Monoclonal antibodies can also be isolated from libraries of phage antibodies using the techniques described in Clackson et al (1991) Nature, 352: 624-628; Marks et al (1991) J. Mol. Biol., 222: 581-597, or of transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr, Opinion 6020 (4): 450-459).
The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a class or subclass of antibody particular, while the rest of the chain (s) is identical or homologous to corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that present the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Nati, Acad. Sci. USA, 81: 6851-6855). Chimeric antibodies include "primatized" antibodies that comprise variable domain antigen binding sequences derived from a non-human primate (e.g., Old World Monkey or Greater Ape) and human constant region sequences.
An "intact antibody" herein is one that comprises a VL and VH domain, as well as a light chain (CL) constant domain and heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be constant domains of native sequence (e.g., constant domains of human native sequence) or variant of the amino acid sequence thereof. The intact antibody can have one or more "effector functions", which refers to those biological activities attributable to the Fe region (a Fe region of native sequence or Fe region variant amino acid sequence) of an antibody. Examples of antibody effector functions include Clq binding; Complement-dependent cytotoxicity; binding to the Fe receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell and BCR receptor.
Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, igG, and IgM, and several of these can be further divided into "subclasses" (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgA, and IgA2 . The constant domains of heavy chains corresponding to the different classes of antibodies are called a, d, e, g, and m, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
Humanization Techniques for reducing the in vivo immunogenicity of a non-human antibody or antibody fragment include so-called "humanization".
A "humanized antibody" refers to a polypeptide comprising at least a portion of a modified variable region of a human antibody wherein a portion of the variable region, preferably a portion substantially smaller than the intact human variable domain, has been substituted with the corresponding sequence from a non-human species and wherein the modified variable region is linked to at least another part of another protein, preferably the constant region of a human antibody. The term "humanized antibodies" includes human antibodies in which one or more amino acid residues of the complementarity determining region ("CDR") and / or one or more amino acid residues of the structural region ("FW" or "FR ") are substituted by amino acid residues from analogous sites in rodents or other non-human antibodies. The term "humanized antibody" also includes a variant of the immunoglobulin amino acid sequence or fragment thereof comprising an FR having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of an immunoglobulin not human The "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. Or, seen in another way, a humanized antibody is a human antibody that also contains selected sequences of non-human (e.g., murine) antibodies in place of the human sequences. A humanized antibody may include conservative amino acid substitutions or unnatural residues of the same species or of different species that do not significantly alter their binding and / or biological activity. Such antibodies are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulins.
There is a range of humanization techniques, including 'CD grafting', 'guided selection', 'deimmunization', 'surface conditioning' (also known as 'inactivation'), 'compound antibodies', 'Optimization of Chain Content' Humana 'and shuffling of structural regions.
CDR graft In this technique, humanized antibodies are human immunoglobulins (receptor antibody) wherein the residues of a complementarity determining region (CDR) of the receptor antibody are substituted by residues of a CDR of a non-human species (donor antibody) such as mouse, rat, camel, bovine, goat or rabbit having the desired properties (in fact, the non-human Care 'grafted' on the human structural region). In some cases, waste from the structural region (FR) of the Human immunoglobulin are substituted by corresponding non-human residues (this can occur when, for example, a particular FR residue has a significant effect on antigen binding).
Additionally, the humanized antibodies may comprise residues that are neither found in the recipient antibody nor in the imported CDR or sequences of the structural region. These modifications are made to refine and further maximize the performance of the antibody. Thus, in general, a humanized antibody will comprise all of at least one, and in one aspect two, variable domains, in which all or all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fe), or that of a human immunoglobulin.
Guided selection The method consists in combining the VH or VL domain of a given specific non-human antibody for a particular epitope with a human VH or VL library and the specific human V domains are selected against the antigen of interest. This selected human VH is combined then with a VL library to generate a fully human VHxVL combination. The method is described in Nature Biotechnology (N.Y.) _12, (1994) 899-903.
Composite antibodies In this method, two or more segments of the amino acid sequence of a human antibody are combined within the final antibody molecule. They are constructed by combining multiple segments of the human VH and VL sequence in combinations that limit or prevent the epitopes of human T cells in the V regions of final composite antibodies. If required, T cell epitopes are limited or prevented by the exchange of segments of the V regions that contribute to or encode a T cell epitope with alternative segments that avoid T cell epitopes. This method is described in US Pat. 2008/0206239 Al.
Deimmunization This method involves the elimination of human T cell epitopes (or another second species) from the V regions of the therapeutic antibody (or other molecule). The sequence of the V regions of therapeutic antibodies is analyzed for the presence of MHC class II binding motifs by, for example, comparison with databases of MHC-binding motifs (such as the database of " reasons "hosted on www .wehi.edu.au).
Alternatively, MHC class II binding motifs can be identified using computational folding methods such as those contemplated by Altuvia et al (J. Mol. Biol.249244-250 (1995)); in these methods, the consecutive overlapping peptides of the V-region sequences are being tested for their MHC class II protein binding energies. These data can then be combined with information on other sequence characteristics that refer to satisfactorily presented peptides, such as amphipathicity, Rothbard motifs, and cleavage sites for cathepsin B and other processing enzymes.
Once T cell (eg, human) epitopes of second potential species have been identified, they are removed by altering one or more amino acids. Modified amino acids are normally within the T cell epitope itself, but may also be adjacent to the epitope in terms of the primary or secondary structure of the protein (and, therefore, may not be adjacent in the primary structure). Most typically, the alteration is by way of substitution but, in some circumstances, the addition or deletion of amino acids will be more appropriate.
All alterations can be made using recombinant DNA technology, so that the The final molecule can be prepared by expressing a recombinant host using well-established methods such as site-directed mutagenesis. However, the use of protein chemistry or any other means of molecular alteration is also possible.
Surface conditioning This method implies: (a) determining the conformational structure of the variable region of the non-human antibody (eg, rodent) (or fragment thereof) by constructing a three-dimensional model of the variable region of the non-human antibody; (b) generate sequence alignments using distributions of relative accessibility from X-ray crystallographic structures of a sufficient number of heavy and light chains of the variable regions of non-human and human antibodies to give a set of positions of the structural regions of heavy and light chains where the alignment positions are identical in 98% of the sufficient number of heavy and light chains of non-human antibodies; (c) define for the non-human antibody that a set of amino acid residues exposed on the surface of the heavy and light chains will be humanized using the set of positions of the region structural generated in step (b); (d) identifying from the amino acid sequences of human antibody a set of amino acid residues exposed on the surface of the heavy and light chains that is most closely identical to the set of amino acid residues exposed on the surface defined in the step (c), wherein the heavy and light chain of the human antibody is or is not naturally paired; (e) substituting, in the amino acid sequence of the non-human antibody to be humanized, the set of amino acid residues exposed on the surface of the heavy and light chains defined in step (c) with the set of amino acid residues exposed on the surface of the heavy and light chains identified in step (d); (f) constructing a three-dimensional model of the variable region of the non-human antibody resulting from the substitution specified in step (e); (g) identify, by comparing the three-dimensional models constructed in steps (a) and (f), any amino acid residue of the sets identified in steps (c) or (d), which are within 5 Angstroms of any atom of any residue of the regions determining the complementarity of the non-human antibody that will be humanized; Y (h) changing any residue identified in step (g) of the original non-human amino acid residue to thereby define a humanizing set of non-human antibodies of amino acid residues exposed on the surface; with the proviso that step (a) does not need to be performed first, but must be done before step (g).
Superhumanization The method compares the non-human sequence with the repertoire of functional human germline genes. Those human genes that encode canonical structures identical or closely related to non-human sequences are selected. The selected human genes with the highest homology within the CDRs are chosen as RF donors. Finally, non-human CDRs are grafted onto these human FRs. This method is described in patent WO 2005/079479 A2.
Content Optimization of the Human Chain This method compares the non-human (for example, mouse) sequence with the repertoire of human germline genes and the differences are scored as Human Chain Content (HSC) that quantifies a sequence in the level of epitopes of MHC / potential T cells. Next, the target sequence is humanized maximizing its HSC instead of using a global identity measurement to generate multiple diverse humanized variants (described in Molecular Immunology, 44, (2007) 1986-1998).
Shuffling of Structural Regions The CDRs of the non-human antibody are fused in frame with cDNA pools encompassing all the structural regions of human germline genes of known heavy and light chains. Next, the humanized antibodies are selected, for example, by in -purposing the antibody library displayed in phage. This is described in Methods 36, 43-60 (2005).
Examples of cell binding agents include those agents described for use in WO 2007/085930, which is incorporated herein.
The following are antigens associated with tumor and related antibodies for use in the embodiments of the present invention.
ANTIGENS ASSOCIATED WITH TUMOR AND RELATED ANTIBODIES (D BMPR1B (bone morph protein receptor type IB bone) Nucleotide Access of GenBank No. NM 001203 Genbank version No. NM 001203.2 GI: 169790809 Update date of the GenBank registry: September 23, 201202: 06 PM Polypeptide Access of GenBank No. NP_001194 Genbank version No. NP_001194.1 GI: 4502431 Date of GenBank registration update: September 23, 201202: 06 PM Cross-references Ten Dijke, P., et al Science 264 (5155): 101-104 (1994), Oncogene 14 (11): 1377-1382 (1997); W02004 / 063362 (Claim 2); W02003 / 042661 (Claim 12); US2003 / 134790-A1 (Pages 38-39); W02002 / 102235 (Claim 13; Page 296); W02003 / 055443 (Page 91-92); WO2002 / 99122 (Example 2; Pages 528-530); W02003 / 029421 (Claim 6); W02003 / 024392 (Claim 2, Fig.112); WO2002 / 98358 (Claim 1; Page 183); W02002 / 54940 (Pages 100-101); WO2002 / 59377 (Pages 349-350); W02002 / 30268 (Claim 27; Page 376); W02001 / 48204 (Example; Fig. 4); NP_001194 receptor for bone morphogenetic protein, type lB / pid = NP_001194.1 .; MIM: 603248; AY065994 (2) The 6 (LATI, SLC7A5) Nucleotide Access of GenBank No. NM 003486 Genbank version No. NM 003486.5 GI: 71979931 Update date of the GenBank registry: June 27, 201212: 06 PM Polypeptide Access of GenBank No. NP_003477 Genbank version No. NP_003477.4 GI: 71979932 Date of GenBank registration update: June 27, 201212: 06 PM Cross-references Biochem. Biophys. Res.
Commun. 255 (2), 283-288 (1999), Nature 395 (6699): 288-291 (1998), Gaugitsch, H.W., et al (1992) J.
Biol. Chem. 267 (16): 11267-11273); W02004 / 048938 (Example 2); WO2004 / 032842 (Example IV); W02003 / 042661 (Claim 12); W02003 / 016475 (Claim 1); WO2002 / 78524 (Example 2); 25 W02002 / 99074 (Claim 19; Pages 127-129); WO2002 / 86443 (Claim 27; Pages 222, 393); W02003 / 003906 (Claim 10; Page 293); WO2002 / 64798 (Claim 33; Pages 93-95); W02000 / 14228 (Claim 5; Pages 133-136); US2003 / 224454 (Fig. 3); W02003 / 025138 (Claim 12; Page 150); NP_003477 family 7 of transporters and solutes (cationic amino acid transporter, and + system), member 5 / pid = NP_003477.3 - Homo sapiens; MIM: 600182; NM_015923. (3) STEAP1 (epithelial antigen of six transmembrane of the prostate) Nucleotide Access of GenBank No. NM_012449 Genbank version No. NM_012449.2 GI: 22027487 GenBank registration update date: September 9, 201202: 57 PM Polypeptide Access of GenBank No. NP_036581 Genbank version No. NP_036581.1 GI: 9558759 Date of GenBank registration update: September 9, 201202: 57 PM Cross-references Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R.S., et al (1999) Proc. Nati Acad. Sci. U. S. A. 96 (25): 14523-14528); W02004 / 065577 (Claim 6); W02004 / 027049 (Fig. 1L); EP1394274 (Example 11); W02004 / 016225 (Claim 2); W02003 / 042661 (Claim 12); US2003 / 157089 (Example 5); US2003 / 185830 (Example 5); US2003 / 064397 (Fig. 2); W02002 / 89747 (Example 5; Pages 618-619); W02003 / 022995 (Example 9, Fig. 13A, Example 53, Page 173, Example 2, Fig. 2A); epithelial antigen of six transmembrane of the prostate; MIM: 604415 (4) 0772P (CAI 25 r MUC16) Nucleotide Access of GenBank No. AF361486 Genbank version No. AF361486.3 GI.-34501466 Date of GenBank registration update: March 11, 201007: 56 AM Polypeptide Access of GenBank No. AAK74120 Genbank version No. AAK74120.3 GI: 34501467 Date of GenBank registration update: March 11, 201007: 56 AM Cross-references J. Biol. Chem. 276 (29): 27371-27375 (2001)); W02004 / 045553 (Claim 14); WO2002 / 92836 (Claim 6, Fig. 12); WO2002 / 83866 (Claim fifteen; Pages 116-121); US2003 / 124140 (Example 16); GI: 34501467; (5) MPF (MPF, MSLN, SMR, megakaryocyte enhancing factor, mesothelin) Nucleotide Access of GenBank No. NM_005823 Genbank version No. NM_005823.5 GI: 293651528 Date of GenBank registration update: September 2, 201201: 47 PM Polypeptide Access of GenBank No. NP_005814 Genbank version No. NP 005814.2 GI: 53988378 Update date of GenBank registration: September 2, 201201: 47 PM Cross-references Yamaguchi, N., et al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Na ti. Acad. Sci. U. S. A. 96 (20): 11531-11536 (1999), Proc. Nati Acad. Sci. U. S. A. 93 (1): 136-140 (1996), J. Biol. Chem. 270 (37): 21984-21990 (1995); W02003 / 101283 (Claim 14); W02002 / 102235 (Claim 13; Pages 287-288); W02002 / 101075 (Claim 4; Pages 308-309); WO2002 / 71928 (Pages 320-321); WO94 / 10312 (Pages 52-57); IM: 601051 (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, family 34 of transporter and solute (sodium phosphate), member 2, phosphate-dependent transporter 3b type ID sodium Nucleotide Access of GenBank No. NM_006424 Genbank version No. NM_006424.2 GI: 110611905 Date of GenBank registration update: July 22, 201203: 39 PM Polypeptide Access of GenBank No. NP_006415 Genbank version No. NP_006415.2 GI: 110611906 GenBank registration update date: July 22, 201203: 39 PM Cross-references J. Biol. Chem. 277 (22): 19665-19672 (2002), Genomics 62 (2): 281-284 (1999), Feild, J.A., et al (1999) Biochem. Bophys. Res. Commun. 258 (3): 578-582); W02004 / 022778 (Claim 2); EP1394274 (Example 11); W02002 / 102235 (Claim 13; Page 20 326); EP0875569 (Claim 1; Pages 17-19); W02001 / 57188 (Claim 20; Page 329); W02004 / 032842 (Example IV); W02001 / 75177 (Claim 24; Pages 139-140); MIM: 604217. (7) Sema 5b (FLJ10372, KIAA1445, Mm. 42015, SEMA5B, SEMAG, sema f urine 5b Hlog, sema 25 domain, seven repeats of thrombospondin (type 1 and similar to type 1), transmembrane domain (TM) and short toplasmic domain ci, (sema f urine) 5B) Nucleotide Access of GenBank No. AB040878 Genbank version No. AB040878.1 GI-.7959148 Date of GenBank registration update: August 2, 200605: 40 PM Polypeptide Access of GenBank No. BAA95969 65 Genbank version No. BAA95969.1 GI: 7959149 Date of GenBank registration update: August 2, 200605: 40 PM Cross-references Nagase T., et al (2000) DNA Res. 7 (2): 143-150); W02004 / 000997 (Claim 1); W02003 / 003984 (Claim 1); W02002 / 06339 (Claim 1; Page 50); W02001 / 88133 (Claim 1; Pages 41-43, 48-58); W02003 / 054152 (Claim 20); W02003 / 1014 00 (Claim 11); Access: 30 Q9P283; Genew; HGNC: 10737 (8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA gene 2700050C12) Nucleotide Access of GenBank No. AY358628 Genbank version No. AY358628.1 GI: 37182377 Date of GenBank registration update: December 1, 200904: 15 AM Polypeptide Access from GenBank No. AAQ88991 Genbank version No. AAQ88991.1 GI: 37182378 Date of GenBank registration update: December 1, 200904: 15 AM Cross-references Ross et al (2002) Cancer Res. 62: 2546-2553; US2003 / 129192 (Claim 2); US2004 / 044180 (Claim 12); US2004 / 044179 35 (Claim 11); US2003 / 096961 (Claim 11); US2003 / 232056 (Example 5); W02003 / 105758 16 (Claim 12); US2003 / 206918 (Example 5); EP1347046 (Claim 1); W02003 / 025148 (Claim 20); GI: 37182378. (9) ETBR (endothelin type B receptor) Nucleotide Access of GenBank No. AY275463 Genbank version No. AY275463.1 GI: 30526094 Date of GenBank registration update: March 11, 201002: 26 AM Polypeptide Access of GenBank No. AAP32295 Genbank version No. AAP32295.1 GI: 30526095 GenBank registration update date: March 11, 201002: 26 AM Cross-references Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991; Ogawa Y., et al Biochem. Biophys. Res.
Commun. 178, 248-255, 1991; Arai H., et al. Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshobagy N.A., et al J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc. Pharmacol. 20, sl-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999; Strausberg R.L., et al Proc.
Nati Acad. Sci. U. S. A. 99, 16899-16903, 2002; Bourgeois C., et al J. Clin. Endocrinol Metab. 82, 3116-3123, 1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R.M.W., et al. Eur. J. Hum. Genet 5, 180-185, 1997; Puffenberger E.G., et al Cell 79, 1257-1266, 1994; Attie T., et al Hum. Mol. Genet 4, 2407-15 2409, 1995; Auricchio A., et al Hum. Mol. Genet 5: 351-354, 1996; Amiel J., et al Hum. Mol.
Genet 5, 355-357, 1996; Hofstra R.M.W., et al Nat. Genet 12, 445-447, 1996; Svensson P.J., et al Hum.
Genet 103, 145-148, 1998; Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002) Hum. Genet 111, 198-206; W02004 / 045516 (Claim 1); W02004 / 048938 (Example 2); W02004 / 040000 (Claim 151); W02003 / 087768 (Claim 1); 20 W02003 / 016475 (Claim 1); W02003 / 016475 (Claim 1); W02002 / 61087 (Fig. 1); W02003 / 016494 (Fig. 6); W02003 / 025138 (Claim 12; Page 144); W02001 / 98351 (Claim 1; Pages 124-125); EP0522868 (Claim 8, Fig.2); W02001 / 77172 (Claim 1; Pages 297-299); US2003 / 109676; US6518404 (Fig. 3); US5773223 (Claim la; Col 31-34); W02004 / 001004. (10) SG783 (RNF124, hypothetical protein FLJ20315) Nucleotide Access of GenBank No. NM 017763 Genbank version No. NM 017763.4 GI: 167830482 Update date of GenBank registration: July 22, 201212: 34 AM Polypeptide Access of GenBank No. NP_060233 Genbank version No. NP_060233.3 GI: 56711322 Date of GenBank registration update: July 22, 201212: 34 AM Cross-references W02003 / 104275 (Claim 1); W02004 / 046342 (Example 2); W02003 / 042661 (Claim 12); W02003 / 083074 (Claim 14; Page 61); W02003 / 018621 (Claim 1); WO2003 / 024392 (Claim 2; Fig. 93); W02001 / 66689 (Example 6); Locus ID: 54894. (11) STEAP2 (HGNC_ 8639, IPCA-1, PCANAP1, STAMP1, STEAP2 STMP 1 gene associated Prostate ta cancer associated protein 1 prostate cancer antigen epi telial six transmembrane Prostate ta 2 protein Prostate six transmembrane ta) Nucleotide Access of GenBank No. AF455138 Genbank version No. AF455138.1 GI: 22655487 Date of GenBank registration update: March 11, 201001: 54 AM Polypeptide Access from GenBank No. AAN04080 Genbank version No. AAN04080.1 GI: 22655488 Date of GenBank registration update: March 11, 201001: 54 AM Cross-references Lab. Invest. 82 (11): 1573-1582 (2002); W02003 / 087306; US2003 / 064397 (Claim 1, Fig. 1); WO2002 / 72596 (Claim 13; Pages 54-55); WO2001 / 72962 (Claim 1, Fig.4B); 35 W02003 / 104270 (Claim 11); W02003 / 104270 (Claim 16); US2004 / 005598 (Claim 22); W02003 / 042661 (Claim 12); US2003 / 060612 (Claim 12, Fig. 10); WO2002 / 26822 (Claim 23, Fig.2); WO2002 / 16429 (Claim 12, Fig.10); GI: 22655488. (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, cationic channel 5 of transient receptor potential, subfamily M, member 4) Nucleotide Access of GenBank No. NM_017636 Genbank version No. NM_017636.3 GI: 304766649 Date of GenBank registration update: June 29, 201211: 27 AM Polypeptide Access of GenBank No. NP 060106 Genbank version No. NP 060106.2 GI: 21314671 Update date of the GenBank registry: June 29, 201211: 27 DM Cross-references Xu, X.Z., et al Proc. Nati Acad. Sel. U. S. A. 98 (19): 10692-10697 (2001), Cell 109 (3): 397-407 (2002), J.
Biol. Chem. 278 (33): 30813-30820 (2003); US2003 / 143557 (Claim 4); W02000 / 40614 (Claim 14; Pages 100-103); W02002 / 10382 (Claim 1, Fig.9A); W02003 / 042661 (Claim 12); WO2002 / 30268 (Claim 27; Page 391); US2003 / 219806 (Claim 4); WO2001 / 62794 (Claim 10 14; Fig. 1A-D); MIM: 606936. (13) CRYPT (CR, CR1, CRGF, CRYPT, TDGF1, growth factor derived from teratocarcinoma) Nucleotide Access of GenBank No. NM_003212 Genbank version No. NM_003212.3 GI: 292494881 Date of GenBank registration update: September 23, 201202: 27 PM Polypeptide Access of GenBank No. NP_8003203 Genbank version No. NP_8003203.1 GI: 4507425 Update date of the GenBank registry: September 23, 201202: 27 PM Cross-references Ciccodicola, A., ET AL EMBO J. 8 (7): 1987-1991 (1989), Am. J. HHumum .. GGeenneett .. 49 (3): 555-565 (1991); US2003 / 224411 (Claim 1); W02003 / 083041 (Example 1); WO2003 / 034984 (Claim 12); W02002 / 88170 (Claim 2; Pages 52-53); W02003 / 024392 (Claim 2, Fig. 58); W02002 / 16413 (Claim 1; Pages 94-95, 105); W02002 / 22808 (Claim 2; Fig. 1); US5854399 (Example 2; Col 17-18); US5792616 (Fig. 2); MIM: 187395 (14) CD21 (CR2 (complement 2 receptor) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792) Nucleotide Access of GenBank No. M26004 Genbank version No. M26004.1 GI: 181939 Date of GenBank registration update: June 23, 201008: 47 AM Polypeptide Access of GenBank No. AAA35786 Genbank version No. AAA35786.1 GI: 181940 Update date of GenBank registration: June 23, 201008: 47 AM Cross-references Fujisaku, et al (1989) J. Biol. Chem. 264 (4): 2118-2125); Weis J.J., et al J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Na ti. Acad. Sci. USES. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35, 1025-1031, 1998; Weis J.J., et al Proc. Nati Acad. Sci.
U. S. A. 83, 5639-5643, 1986; Sinha S.K., et al (1993) J.
Immunol. 150, 5311-5320; W02004 / 045520 (Example 4); US2004 / 005538 (Example 1); W02003 / 062401 (Claim 9); WO2004 / 045520 (Example 4); WO91 / 02536 (Fig. 9.1-9.9); W02004 / 020595 (Claim 1); Access: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1. (15) CD79b (CD79B, CD793, IGb (beta associated with immunoglobulin), B29) Nucleotide Access of GenBank No. NM_000626 Genbank version No. NM_000626.2 GI: 90193589 GenBank registration update date: June 26, 201201: 53 PM Polypeptide Access of GenBank No. NP_000617 Genbank version No. NP_000617.1 GI: 11038674 Date of GenBank registration update: June 26, 201201: 53 PM Cross-references Proc. Nati Acad. Sci. U. S. A. (2003) 100 (7): 4126-4131, Blood (2002) 100 (9): 3068-3076, Muller et al. (1992) Eur. J. Immunol. 22 (6): 1621-1625); W02004 / 016225 (Claim 2, Fig.140); W02003 / 087768, US2004 / 101874 (Claim 1, Page 102); W02003 / 062401 (Claim 9); WO2002 / 78524 (Example 2); US2002 / 150573 (Claim 355, Page 15); US5644033; W02003 / 048202 (Claim 1, Pages 306 and 309); WO 99/58658, US6534482 (Claim 13, Fig. 17A / B); W02000 / 55351 (Claim 11, Pages 1145-1146); MIM: 147245 (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain that contains anchor protein f fsa f atasa la 5), SPAP1B, SPAP1C) Nucleotide Access of Genbank No. NM_030764 Genbank version No. NM_030764.3 GI: 227430280 Date of GenBank registration update: June 30, 201212: 30 AM Polypeptide Access of Genbank No. NP_110391 Genbank version No. NP_110391.2 GI: 19923629 Update date of the GenBank registry: June 30, 201212: 30 AM Cross-references AY358130); Genome Res. 13 (10): 2265-2270 (2003), Immunogenetics 54 (2): 87-95 (2002), Blood 99 (8): 2662-2669 (2002), Proc. Na ti. Acad. Sci. U. S A. 98 (17): 9772-9777 (2001), Xu, M.J., et al (2001) Biochem. Biophys. Res. Commun. 280 (3): 768-775; W0200 / 016225 (Claim 2); W02003 / 077836; W02001 / 38490 (Claim 5; Fig.18D-1- 18D-2); W02003 / O97803 (Claim 12); 10 W02003 / 089624 (Claim 25); MIM: 606509. (17) HER2 (ErbB2) Nucleotide Access of Genbank No. M11730 Genbank version No. M11730.1 GI: 183986 Date of GenBank registration update: June 23, 201008: 47 AM Polypeptide Access of Genbank No. AAA75493 Genbank version No. AAA75493.1 GI: 306840 Update date of GenBank registration: June 23, 201008: 47 AM Cross-references Coussens L., et al Science (1985) 230 (4730): 1132-1139); Yamamoto T., et al Nature 319, 230-234, 1986; Semba K., et al Proc. Nati Acad. Sci. U. S.A. 82, 6497-6501, 1985; Swiercz J.M., et al J. Cell Biol. 165, 869-15880, 2004; Kuhns J.J., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993) Genomics 15, 426-429; W02004 / 048938 (Example 2); W02004 / 027049 (Fig. II); W02004 / 009622; W02003 / 081210; W02003 / 089904 (Claim 9); W02003 / 016475 (Claim US2003 / 118592; W02003 / 008537 (Claim 1); W02003 / 055439 (Claim 29; Fig. 1A-B); WO2003 / 025228 (Claim 37, Fig. 5C); WO2002 / 22636 (Example 13; page 95-107); W02002 / 12341 (Claim 68, Fig.7); WO2002 / 13847 (Pages 71-74); W02002 / 14503 (Pages 114-117); WO2001 / 53463 (Claim 2; Pages 41-46); W02001 / 41787 (Page 15); W02000 / 44899 (Claim 52, Fig.7); W02000 / 20579 (Claim 3, Fig. 2); US5869445 (Claim 3; Col 31-38); WO9630514 (Claim 2; Pages 56-61); EP1439393 (Claim 7); W02004 / 043361 (Claim 7); W02004 / 022709; W02001 / 00244 25 (Example 3, Fig. 4); Access: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1 ANTIBODIES Abbott: US20110177095 For example, an antibody comprising CDRs that generally have at least 80% sequence identity with CDRs having amino acid sequences of SEQ ID NO: 3 (CDR-HI), SEQ ID NO: 4 (CDR-H2) , SEQ ID NO: 5 (CDR-H3), SEQ ID NO: 104 and / or SEQ ID NO: 6 (CDR-L1), SEQ ID NO: 7 (CDR-L2), and SEQ ID NO: 8 (CDR -L3), wherein the anti-HER2 antibody or anti-HER2 binding fragment has reduced immunogenicity compared to an antibody having a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 2.
Biogen: US20100119511 For example, ATCC access numbers: PTA-10355, PTA-10356, PTA-10357, PTA-10358 For example, a purified antibody molecule that binds HER2 comprising all six CDRs of an antibody selected from the group consisting of BIIB71F10 (SEQ ID NOs: 11, 13), BIIB69A09 (SEQ ID NOs: 15, 17); BIIB67F10 (SEQ ID NOs: 19, 21); BIIB67F11 (SEQ ID NOs: 23, 25), BIIB66A12 (SEQ ID NOs: 27, 29), BIIB66C01 (SEQ ID NOs: 31, 33), BIIB65C10 (SEQ ID NOs: 35, 37), BIIB65H09 (SEQ ID NOs: 39, 41) and BIIB65B03 (SEQ ID NOs: 43, 45), or CDRs that are identical or have no more than two alterations of said CDRs.
Herceptin (Genentech) -US6,054,297; do not. of access of ATCC CRL-10463 (Genentech) Pertuzumab (Genentech) US20110117097 for example, see SEQ ID NOs: 15 and 16, SEC ID NOs: 17 and 18, SEQ ID NOs: 23 and 24 and ATCC accession numbers HB-12215, HB-12216, CRL 10463, HB-12697.
US20090285837 US20090202546 for example, ATCC access numbers: HB-12215, HB-12216, CRL 10463, HB-12698.
US20060088523 for example, ATCC access numbers: HB- 12215, HB-12216 - for example, an antibody comprising variable light and heavy variable amino acid sequences in SEQ ID NOs: 3 and 4, respectively. - for example, an antibody comprising an amino acid sequence of the light chain selected from SEQ ID NOs: 15 and 23 and a heavy chain amino acid sequence selected from SEQ ID NOs: 16 and 24 US20060018899 - for example, ATCC access numbers: (7C2) HB-12215, (7F3) HB-12216, (4D5) CRL-10463, (2C4) HB-12697. - for example, an antibody comprising the amino acid sequence in SEQ ID NO: 23, or a deamidated and / or oxidized variant thereof.
US2011 / 0159014 - for example, an antibody having a light chain variable domain comprising the hypervariable regions of SEQ ID NO: 1.
- For example, an antibody having a heavy chain variable domain comprising the hypervariable regions of SEQ ID NO: 2.
US20090187007 Glycotope: TrasGEX antibody http://www.glycotope.com/pipeline For example, see International Joint Cancer Institute and Changhai Cancer Center Hospital: HMTI-Fc Ab-Gao J., et al BMB Rep. Oct 31, 2009; 42 (10): 636-41.
Symphogen: US20110217305 Union Stem Cell & Gene Engineering, China - Liu HQ., Et al Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. May 2010; 26 (5): 456-8. (18) NCA (CEACAM6) Nucleotide Access of Genbank No. M18728 Genbank version No. M18728.1 GI: 189084 Date of GenBank registration update: June 23, 201008: 48 AM Polypeptide Access of Genbank No. AAA59907 Genbank version No. AAA59907.1 GI: 189085 Update date of the GenBank registration: June 23, 201008: 48 AM Cross-references Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R.L., et al Proc. Nati Acad. Sel.
U. S. A. 99: 16899-16903, 2002; W02004 / 063709; EP1439393 (Claim 7); W02004 / 044178 (Example 4); W02004 / 031238; W02003 / 042661 (Claim 12); WO2002 / 78524 (Example 2); WO2002 / 86443 (Claim 27; Page 427); W02002 / 60317 (Claim 2); Access: P40199; Q14920; EMBL; M29541; AAA59915.1.
EMBL; M18728. (19) MDP (DPEPD Nucleotide Access of Genbank No. BC017023 Genbank version No. BC017023.1 GI: 16877538 Date of GenBank registration update: March 6, 201201: 00 PM Polypeptide Access of Genbank No. AAH17023 Genbank version No. AAH17023.1 GI: 16877539 Date of GenBank registration update: March 6, 201201: 00 PM Cross-references Proc. Nati Acad. Sci. U. S. A. 99 (26): 16899-16903 (2002); W02003 / 016475 (Claim 1); WO2002 / 64798 (Claim 33; Page S 85-87); JP05003790 (Fig.6-8); W099 / 46284 (Fig.9); MIM: 179780 (20) IL20R-alpha (IL20Ra ZCYTOR7) Nucleotide Access of Genbank No. AF184971 Genbank version No. AF184971.1 GI: 6013324 Update date of the GenBank registry: March 10, 201010: 00 PM Polypeptide Access of Genbank No. AAF01320 Genbank version No. AAF01320.1 GI: 6013325 Date of GenBank registration update: March 10, 201010: 00 PM Cross-references Clark H.F., et al Genome Res. 13, 2265-2270, 2003; Mungall A.J., et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001; Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003) 10 Biochemistry 42: 12617-12624; Sheikh F., et al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004 / 005320 (Example 5); W02003 / 029262 (Pages 74-75); W02003 / 002717 (Claim 2; Page 63); WO2002 / 22153 (Pages 45-47); US2002 / 042366 (Pages 20-21); W02001 / 46261 (Pages 57-59); W02001 / 46232 (Pages 63-65); W098 / 37193 (Claim 1; Pages 55-59); Access: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1. (21) B revi can (BCAN, BEHAB) Nucleotide Access of Genbank No. AF229053 Genbank version No. AF229053.1 GI: 10798902 GenBank registration update date: March 11, 201012: 58 AM Polypeptide Access of Genbank No. AAG23135 Genbank version No. AAG23135.1 GI: 10798903 Date of GenBank registration update: March 11, 201012: 58 AM Cross-references Gary S.C., et al Gene 256, 139-147, 2000; Clark H.F., et al Genome Res. 13, 2265-2270, 2003; Strausberg R.L., et al Proc. Nati Acad. Scí. U. S. A. 99, 16899-16903, 2002; US2003 / 186372 (Claim 11); US2003 / 186373 (Claim 11); US2003 / 119131 (Claim 1, Fig. 52); US2003 / 119122 (Claim 1; 20 Fig. 52); US2003 / 119126 (Claim 1); US2003 / 119121 (Claim 1, Fig.52); US2003 / 119129 (Claim 1); US2003 / 119130 (Claim 1); US2003 / 119128 (Claim 1, Fig.52); US2003 / 119125 (Claim 1); W02003 / 016475 (Claim 1); W02002 / 02634 (Claim 1) (22) EphB2R (DRT, ERK, Hek5, EPHT3, TyroS) Nucleotide Access of Genbank No. NM_004442 Genbank version No. NM_004442.6 GI: 111118979 Date of GenBank registration update: September 8, 201204: 43 PM Polypeptide Access of Genbank No. NP_004433 Genbank version No. NP_004433.2 GI: 21396504 Date of GenBank registration update: September 8, 201204: 43 PM Cross-references Chan, J. and Watt, V.M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5): 897-905 (1995), Annu. Rev. Neurosci. 21: 309-345 (1998), Int. Rev. Cytol. 196: 177-244 (2000); W02003042661 (Claim 12); W0200053216 (Claim 1; Page 41); W02004065576 (Claim 1); W02004020583 (Claim 9); W02003004529 (Pages 128-132); W0200053216 (Claim 1; Page 42); MIM: 600997 (23) ASLG659 (B7h) Nucleotide Access of Genbank No. AX092328 Genbank version No. AX092328.1 GI: 13444478 Date of GenBank registration update: January 26, 201107: 37 AM Cross-references US2004 / 0101899 (Claim 2); W02003104399 (Claim 11); W02004000221 (Fig. 3); US2003 / 165504 (Claim 1); US2003 / 124140 (Example 2); US2003 / 065143 (Fig. 60); W02002 / 102235 (Claim 13; Page 299); US2003 / 091580 (Example 2); W02002 / 10187 (Claim 6, Fig. 10); WO2001 / 94641 (Claim 12; Fig.7b); W02002 / 02624 (Claim 13, Fig.1A-1B); US2002 / 034749 (Claim 54; Pages 45-46); W02002 / 06317 (Example 2; Pages 320-321, Claim 34; Pages 321-322); WO2002 / 71928 (Pages 468-469); W02002 / 02587 (Example 1, Fig.1); W02001 / 40269 (Example 3; Pages 190-192); W02000 / 36107 (Example 2; pages 205-207); W02004 / 053079 (Claim 12); W02003 / 004989 (Claim 1); WO2002 / 71928 (Pages 233-234, 452-453); WO 01/16318. (24) PSCA (Precursor of cytoblastic antigens of the prostate) Nucleotide Access of Genbank No. AJ297436 Genbank version No. AJ297436.1 GI: 9367211 GenBank registration update date: February 1, 201111: 25 AM Polypeptide Access of Genbank No. CAB97347 Genbank version No. CAB97347.1 GI: 9367212 Date of GenBank registration update: February 1, 201111: 25 AM Cross-references Reiter R.E., et al Proc. Nati Acad. Sci. USES. 95, 1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275 (3): 783-788; W02004 / 022709; EP1394274 (Example 11); US2004 / 018553 (Claim 17); W02003 / 008537 (Claim 1); WO2002 / 81646 (Claim 1; Page 164); W02003 / 003906 (Claim 10, page 288); W02001 / 40309 (Example 1, Fig. 17); US2001 / 055751 (Example 1; Fig. Ib); W02000 / 32752 (Claim 18, Fig. 1); WO98 / 51805 (Claim 17; Page 97); W098 / 51824 (Claim 10; Page 94); W098 / 40403 (Claim 2, Fig. IB); Access: 043653; EMBL; AF043498; AAC39607.1 (25) GE DA Nucleotide Access of Genbank No. AY260763 Genbank version No. AY260763.1 GI: 30102448 GenBank registration update date: March 11, 201002: 24 AM Polypeptide Access of Genbank No. AAP14954 Genbank version No. AAP14954.1 GI: 30102449 Date of GenBank registration update: March 11, 201002: 24 AM Cross-references Protein similar to lipoma fusion pattern HMGIC AP14954 / pid = AAP14954 .1 - Homo sapiens (human); W02003 / 054152 (Claim 20); W02003 / 000842 (Claim 1); W02003 / 023013 (Example 3, Claim 20); US2003 / 194704 (Claim 45); GI: 30102449. (26) BAFF-R (receptor for B cell activating factor, receptor 3 of BLyS, BR3) Nucleotide Access of Genbank No. AF116456 Genbank version No. AF116456.1 GI: 4585274 Date of GenBank registration update: March 10, 201009: 44 PM Polypeptide Access of Genbank No. AAD25356 Genbank version No. AAD25356.1 GI: 4585275 Date of GenBank registration update: March 10, 201009: 44 PM Cross-references BAFF Receptor / pid = NP_443177.1 - Homo sapiens: Thompson, J.S., et al Science 293 (5537), 2108-2111 (2001); W02004 / 058309; WO2004 / 011611; W02003 / 045422 (Example; Pages 32-33); W02003 / 014294 (Claim 35, Fig.6B); W02003 / 035846 (Claim 70; Pages 615-616); WO2002 / 94852 (Col 136-137); WO2002 / 3876625 (Claim 3; Page 133); W02002 / 24909 (Example 3, Fig. 3); MIM: 606269; NP 443177.1; NM 0529451; AF132600 (27) CD22 (Isoform of the CD22-B receptor of B cells, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814) Nucleotide Access of Genbank No. AK026467 Genbank version No. AK026467.1 GI: 10439337 GenBank registration update date: September 11, 200611: 24 PM Polypeptide Access of Genbank No. BAB15489 Genbank version No. BAB15489.1 GI: 10439338 Date of GenBank registration update: September 11, 200611: 24 PM Cross-references Wilson et al (1991) J. Exp. Med. 173: 137-146; W02003 / 072036 (Claim 1, Fig. 1); IM: 107266; NP_001762.1; NM_001771_1. (27a) CD22 (Molecule CD22) Nucleotide Access of Genbank No. X52785 Genbank version No. X52785.1 GI: 29778 GenBank registration update date: February 2, 201110: 09 AM Polypeptide Access of Genbank No. CAA36988 Genbank version No. CAA36988.1 GI: 29779 Update date of GenBank registration: February 2, 201110: 09 AM Cross-references Stamenkovic I. et al., Nature 345 (6270), 74-77 (1990)?? Other information Official symbol: CD22 Other Pseudonyms: SIGLEC-2, SIGLEC2 Other Designations: CD22 B-cell receptor; adhesion molecule to B lymphocyte cells; BL-CAM; CD22 antigen; Leu-14 T cell surface antigen; lectin 2 similar to Ig binding to sialic acid; lectin 2 similar to Ig binding to sialic acid ANTIBODIES G5 / 44 (Inotuzumab): DiJoseph JF., Et al C ncer Immunol Immunother. January 2005; 54 (l): ll-24.
Epratuzu ab - Goldenberg DM., Et al Expe rt Rev Anticancer Ther. 6 (10): 1341-53, 2006. (28) CD79a (CD79A, CD79alpha), alpha associated with immunoglobulin, a B-cell-specific protein that interacts covalently with Ig beta (CD79B) and forms a complex on the surface with IgM molecules, translates a signal that participates in the B cell differentiation), pl: 4. 84, MW: 25028 TM: 2 Chromosome of the Gene [P]: 19ql3.2).
Nucleotide Access of Genbank No. NM_001783 Genbank version No. NM_001783.3 GI: 90193587 GenBank registration update date: June 26, 201201: 48 PM Polypeptide Access of Genbank No. NP_001774 Genbank version No. NP_001774.1 GI: 4502685 Date of GenBank registration update: June 26, 201201: 48 PM Cross-references W02003 / 088808, US2003 / 0228319; W02003 / 062401 (Claim 9); US2002 / 150573 (Claim 4, Pages 13-14); W099 / 58658 (Claim 13, Fig. 16); WO92 / 07574 (Fig. 1); US5644033; Ha, et al (1992) J.
Immunol. 148 (5): 1526-1531; Müller et al (1992) Eur. J. Immunol. 22: 1621-1625; Hashimoto et al (1994) Immunogenetics 40 (4): 287-295; Preud'homme et al (1992) Clin. Exp. 5 Immunol. 90 (1): 141-146; Yu et al (1992) J. Immunol. 148 (2) 633-637; Sakaguchi et al (1988) EMBO J.7 (11): 3457-3464 (29) CXCR5 (Burkitt's Lymphoma Receptor 1, a G-protein coupled receptor that is activated by the chemokine CXCL13, functions in the migration of lymphocytes and humoral defense, plays a role in infection 10 for HIV-2 and perhaps development of AIDS, lymphoma, myeloma and leukemia); 312 aa, pl: 8. 54 MW: 41959 TM: 7 Gen Chromosome [P]: llq23. 3 Nucleotide Access of Genbank No. NM_001716 Genbank version No. NM_001716.4 GI: 342307092 Date of GenBank registration update: September 30, 201201: 49 PM Polypeptide Access of Genbank No. NP_001707 Genbank version No. NP_001707.1 GI: 4502415 Date of GenBank registration update: September 30, 201201: 49 PM Cross-references W02004 / 040000; WO2004 / 015426; US2003 / 105292 (Example 2); US6555339 (Example 2); W02002 / 61087 (Fig.1); W02001 / 57188 (Claim 20, Page 269); W02001 / 72830 (pages 12-13); W02000 / 22129 (Example 1, Pages 152-153, 15 Example 2, pages 254-256); W099 / 28468 (Claim 1, Page 38); US5440021 (Example 2, col 49-52); W094 / 28931 (Pages 56-58); W092 / 17497 (Claim 7, Fig. 5); Dobner et al (1992) Eur. J. Immunol. 22: 2195-2199; Barella et al (1995) Biochem. J. 309: 773-779 (30) HLA-DOB (beta subunit of the MHC class II molecule (antigen la) that binds to peptides and presents to CD4 + T lymphocytes); 273 aa, pl: b, 56, MW: 30820. TM: 1 Gen Chromosome [P]: 6p21. 3) Nucleotide Access of Genbank No. NM_002120 Genbank version No. NM_002120.3 GI: 118402587 Date of GenBank registration update: September 8, 201204: 46 PM Polypeptide Access of Genbank No. NP_002111 Genbank version No. NP_002111.1 GI: 4504403 Date of GenBank registration update: September 8, 201204: 46 PM Cross-references Tonnelle et al (1985) EMBO J. 4 (11): 2839-2847; Jonsson et al (1989) Immunogenetics 29 (6): 411-413; Beck et al (1992) J. Mol. Biol. 228: 433-441; Strausberg et al (2002) Proc. Nati Acad. Sci USA 99: 16899-16903; Servenius et al (1987) J. Biol. Chem. 262: 8759-8766; Beck et al (1996) J. Mol. Biol. 25 255: 1-13; Naruse et al (2002) Tissue Antigens 59: 512-519; W099 / 58658 (Claim 13, Fig. 15); US6153408 (Col 35-38); US5976551 (col 168-170); US6011146 (col 145-146); Kasahara et al (1989) Immunogenetics 30 (1): 66-68; Larham et al (1985) J. Biol. Chem. 260 (26): 14111-14119 (3D P2X5 (Channel 5 ion regulated by the ligand of the P2X purinergic receptor, an ion channel regulated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa, pl: 7. 63, MW: 47206, TM: 1 Gen Chromosome [P]: 17pl3.3).
Nucleotide Access of Genbank No. NM_002561 Genbank version No. NM_002561.3 GI: 325197202 Date of GenBank registration update: June 27, 201212: 41 AM Polypeptide Access of Genbank No. NP_002552 Genbank version No. NP_002552.2 GI: 28416933 Date of GenBank registration update: June 27, 201212: 41 AM Cross-references Le et al (1997) FEBS Lett. 418 (1-2): 195-199; W02004 / 047749; W02003 / 072035 (Claim 10); Touchman et al (2000) Genome Res. 10: 165-173; W02002 / 22660 (Claim 20); W02003 / 093444 (Claim 1); WO2003 / 087768 (Claim 1); W02003 / 029277 (Page 82) (32) CD72 (CD72 B cell differentiation antigen, Lyb-2); 359 aa, pl: 8. 66, MW: 40225, TM: 1 5 Chromosome 65 of the gene [P]: 9pl3. 3) .
Nucleotide Access of Genbank No. NM_001782 Genbank version No. NM_001782.2 GI: 194018444 Date of GenBank registration update: June 26, 201201: 43 PM Polypeptide Access of Genbank No. NP_001773 10 Genbank version No. NP_001773.1 GI: 4502683 GenBank registration update date: June 26, 201201: 43 PM Cross-references W02004042346 (Claim 65); W02003 / 02693 (Pages 51-52, 57-58); W02000 / 75655 (pages 105-106); Von Hoegen et al (1990) J. Immunol. 144 (12): 4870-4877; Strausberg et al (2002) Proc. Nati Acad. Sci USA 99: 16899-16903. (33) LY64 (lymphocyte antigen 64 (RP105), type I membrane protein from the leucine-rich repeat family (LRR), regulates B cell activation and apoptosis, loss of function is associated with elevated disease activity in patients with systemic mucosal lupus erythema); 661 aa, pl: 6.20, MW: 74147 TM: 1 Chromosome of the Gene [P]: 5ql2).
Nucleotide Access of Genbank No. NM 005582 Genbank version No. NM_005582.2 GI: 167555126 Date of GenBank registration update: September 2, 201201: 50 PM Polypeptide Access of Genbank No. NP_005573 Genbank version No. NP_005573.2 GI: 167555127 Date of GenBank registration update: September 2, 201201: 50 PM Cross-references US2002 / 193567; WO97 / 07198 (Claim 11, pages 39-42); Miura et al (1996) 15 Genomics 38 (3): 299- 304; Miura et al (1998) Blood 92: 2815-2822; W02003 / 083047; W097 / 44452 (Claim 8, pages 57-61); W02000 / 12130 (pages 24-26). (34) FcRHl (Protein 1 similar to the Fe receptor, a putative receptor for the immunoglobulin Fe domain containing Ig-like C2-like domains and ITAM, may have a role in the differentiation of B cells); 429 aa, pl: 5.28, MW: 46925 TM: 1 Gen Chromosome [P]: Iq21-lq22) Nucleotide Access of Genbank No. NM_052938 Genbank version No. NM_052938.4 GI: 226958543 Update date of GenBank registration: September 2, 201201: 43 PM Polypeptide Access of Genbank No. NP_443170 Genbank version No. NP_443170.1 GI: 16418419 Date of GenBank registration update: September 2, 201201: 43 PM Cross-references W02003 / 077836; W02001 / 38490 (Claim 6, Fig. 18E-1-18-E-2); Davis et al (2001) Proc. Nati Acad. Sci USA 98 (17): 9772-9777; W02003 / 089624 (Claim 8); EP1347046 (Claim 1); W02003 / 089624 (Claim 7). (35) IRTA2 (2) associated with the translocation of receptors of the super immunoglobulin family, a putative immunoreceptor with possible functions in the development and lymphomagenesis of B cells, the deregulation of the translocalization gene occurs in some malignant B-cell tumors ); 977 aa, pl: 6. 88, MW: 106468, TM: 1 Gene Chromosome [P]: lq21) Nucleotide Access of Genbank No. AF343662 Genbank version No. AF343662.1 GI: 13591709 Date of GenBank registration update: March 11, 201001: 16 AM Polypeptide Access of Genbank No. AAK31325 Genbank version No. AAK31325.1 GI: 13591710 Update date of GenBank registration: March 11, 201001: 16 AM Cross-references AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse: AK089756, AY158090, AY 506558; NP_112571.1; W02003 / 024392 (Claim 2, Fig.97); Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277 (1): 124-127; W02003 / 077836; W02001 / 38490 (claim 3, Fig. 18B-1-18B-2). (36) TENB2 (TMEFF2, tomorregulin, TPEF, HPP1, TR, proteoglycan 35 of transmembrane putative r related to the EGF / heregulin family of growth factors and follistatin); 374 aa) Nucleotide Access of Genbank No. AF179274 Genbank version No. AF179274.2 GI: 12280939 Date of GenBank registration update: March 11, 201001: 05 AM Polypeptide Access of Genbank No. AAD55776 Genbank version No. AAD55776.2 GI: 12280940 Update date of the GenBank registry: March 11, 201001: 05 AM Cross-references NCBI Access: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; AY358907, CAF85723, CQ782436; WO2004 / 074320; JP200 113151; W02003 / 042661; W02003 / 009814; EP1295944 (pages 69-70); W02002 / 30268 (page 329); WO2001 / 90304; US2004 / 249130; US2004 / 022727; W02004 / 063355; US2004 / 197325; US2003 / 232350; 5 US2004 / 005563; US2003 / 124579; Horie et al (2000) Genomics 67: 146-152; Uchida et al (1999) Bíochem. Biophys. Res. Commun. 266: 593-602; Liang et al (2000) Cancer Res. 60: 4907-12; Glynne-Jones et al (2001) Int J Cancer. Oct 15; 94 (2): 178-84. (37) PSMA - FOLH1 (Folate hydrolase (prostate specific membrane antigen) 1) Nucleotide Access of Genbank No. M99487 Genbank version No. M99487.1 GI: 190663 Date of GenBank registration update: June 23, 201008: 48 AM Polypeptide Access of Genbank No. AAA60209 Genbank version No. AAA60209.1 GI: 190664 Update date of the GenBank registration: June 23, 201008: 48 AM Cross-references Israeli R.S. et al Cancer Res. 53 (2), 227-230 (1993) Other information Official Symbol: FOLH1 Other Pseudonyms: GIG27, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdasa, PSM, PSMA, mGCP Other Designations: dipeptidase 1 acid bound to N-acetylated alpha; dipeptidase I acid bound to 5 alpha N-acetylated; NAALADase I; gene 27 protein inhibitor cell growth; folilpoli-gamma-glutamate carboxypeptidase; glutamate carboxylase II; gluta-ato carboxypeptidase 2; glutamate carboxypeptidase II; membrane carboxypeptidase glutamate; F variant of the specific membrane antigen of the prostate; pteroilpoli-gam a-glutamate carboxypeptidase ANTIBODIES US 7,666,425: Antibodies produced by Hybridomas having the following ATCC references: ATCC Accession No. HB-12101, ATCC Accession No. HB-12109, ATCC Accession No. HB-12127 and ATCC Accession No. HB- 12126.
Prosean: a monoclonal antibody selected from the group consisting of 8H12, 3E11, 17G1, 29B4, 30C1 and 20F2 (US 7,811,564; Moffett S., et al Hybridoma (Larchmt), Dec 2007; 26 (6): 363-72).
Cytogen: monoclonal antibodies 7E11-C5 (No.
ATCC Access HB 10494) and 9H10-D4 (Accession No. ATCC HB11430) - US 5,763,202 GlycoMimetics: NUH2 - Access No. of ATCC HB 9762 (US 7,135,301) Human Genome Science: HPRAJ70 - Accession No. of ATCC 97131 (US 6,824,993); amino acid sequence encoded by the cDNA clone (HPRAJ70) deposited as Deposit No. 97131 of the American Type Culture Collection ("ATCC").
Medarex: Anti-PSMA Antibodies Lacking Fucosyl Residues - US 7,875,278 Anti-mouse PSMA antibodies include antibodies 3F5.4G6, 3D7.1.1, 4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6, 4C8B9, and monoclonal. Hybridomas secreting 3F5.4G6, 3D7.1.1, 4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6 or 4C8B9 have been publicly deposited and described in the U.S. Patent No. 6,159,508. Relevant hybridomas have been publicly deposited and are described in U.S. Patent No. 6,107,090. In addition, humanized anti-PSMA antibodies, which include a humanized version of J591, are described in more detail in PCT Publication WO 02/098897.
Other mouse anti-human PSMA antibodies have been described in the art, such as mAb 107-1A4 (Wang, S. et al (2001) Int. J. Cancer 92: 871-876) and mAb 2C9 (Kato, K. Et al (2003) Int. J. Urol.10: 439-444).
Examples of human anti-PSMA monoclonal antibodies include antibodies 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3, isolated and structurally characterized as originally described in PCT Publications WO 01/09192 and WO 03 / 064606 and in the US Provisional Application Serial No. 60 / 654,125, entitled "Human Monoclonal Antibodies to Prostate Specific Membrane Antigen (PSMA)", filed on February 18, 2005. The VH amino acid sequences of 4A3, 7F12, 8C12 , 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 are shown in SEQ ID NOs: 1-9, respectively. The VL amino acid sequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 are shown in SEQ ID NOs: 10-18, respectively.
Other human anti-PSMA antibodies include the antibodies disclosed in PCT Publication WO 03/034903 and North American Application No.2004 / 0033229.
NW Biotherapeutics: A hybridoma cell line selected from the group consisting of 3F5.4G6 having accession number of ATCC HB12060, 3D7-1.I. which has ATCC access number HB12309, 4E10-1.14 that has ATCC access number HB12310, 3E11 (ATCC HB12488), 4D8 (ATCC HB12487), 3E6 (ATCC HB12486), 3C9 (ATCC HB12484), 2C7 (ATCC HB12490), 1G3 (ATCC HB12489), 3C4 (ATCC HB12494), 3C6 (ATCC HB12491), 4D4 (ATCC HB12493), 1G9 (ATCC HB12495), 5C8B9 (ATCC HB12492) and 3G6 (ATCC HB12485) - see US 6,150,508 PSMA Development Company / Progenics / Cytogen Seattle Genetics: mAb 3.9, produced by the hybridoma deposited under ATCC Accession No. PTA-3258 or mAb 10.3, produced by the hybridoma deposited under ATCC Accession No. PTA-3347 - US 7,850,971 PSMA Development Company - Compositions of PSMA antibodies (US 20080286284, Table 1) This application is a divisional of the US patent application Serial No. 10 / 395,894, filed on March 21, 2003 (US 7,850,971) University Hospital of Freiburg, Germany mAbs 3 / A12, 3 / E7, and 3 / F11 (Wolf P., et al Prostate.
Apr 1; 70 (5): 562-9). (38) SST (Somatostatin receptor, note that there are 5 subtypes) (38.1) SSTR2 (Somatostatin Receptor 2) Nucleotide Access of Genbank No. NM_001050 Genbank version No. NMR_001050.2 GI: 44890054 Date of GenBank registration update: August 19, 201201: 37 PM Polypeptide Access of Genbank No. NP_001041 Genbank version No. NP_001041.1 GI: 4557859 Date of GenBank registration update: August 19, 201201: 37 PM Cross-references Yamada Y., et al Proc. Nati Acad. Sci. U.S.A. 89 (1), 251-255 (1992); Susini C., et al Ann Oncol. 2006 Dec; 17 (12): 1733-42 Other information Official Symbol: SSTR2 Other Designations: SRIF-1; SS2R; Somatostatin receptor type 2 (38.2) SSTR5 (Somatostatin Receptor 5) Nucleotide Access of Genbank No. D16827 Genbank version No. D16827.1 GI: 487683 Date of GenBank registration update: August 1, 200612: 45 PM Polypeptide Access of Genbank No. BAA04107 Genbank version No. BAA04107.1 GI: 487684 Update date of GenBank registration: August 1, 200612: 45 PM Cross-references Yamada, Y., et al Biochem. Biophys. Res. Commun. 195 (2), 844-852 (1993) Other information Official Symbol: SSTR5 Other Pseudonyms: SS-5-R Other Designations: Somatostatin receptor subtype 5; Somatostatin receptor type 5 (38.3) SSTR1 (38.4) SSTR3 (38.5) SSTR4 AvB6 ~ Both subunits (39 + 40) (39) ITGAV (Integrin, alpha V; Nucleotide Access of Genbank No. M14648 J02826 M18365 Genbank version No. M14648.1 GI: 340306 Date of GenBank registration update: June 23, 201008: 56 AM Polypeptide Access of Genbank No. AAA36808 Genbank version No. AAA36808.1 GI: 340307 Update date of GenBank registration: June 3, 201008: 56 AM Cross-references Suzuki S., et al Proc. Na ti. Acad. Sci. U. S. A. 83 (22), 8614-8618 (1986) Other information Official Symbol: ITGAV Other Pseudonyms: CD51, MSK8, VNRA, VTNR Other Designations: antigen identified by the monoclonal antibody L230; alpha-V integrin; integrin alfaVbeta3; integrin, alpha V (vitronectin receptor, alpha polypeptide, CD51 antigen); alpha subunit of the vitronectin receptor (40) ITGB6 (Integrin, beta 6) Nucleotide Access of Genbank No. NM_000888 Genbank version No. NM_000888.3 GI: 9966771 Date of GenBank registration update: June 27, 201212: 46 AM Polypeptide Access of Genbank No. NP_000879 Genbank version No. NP_000879.2 GI: 9625002 Date of GenBank registration update: June 27, 201212: 46 AM Cross-references Sheppard D.J., et al Biol. Chem. 265 (20), 11502-11507 (1990) Other information Official Symbol: ITGB6 Other Designations: integrin beta-6 ANTIBODIES Biogen: US 7,943,742 - Hybridoma clones 6. 3G9 and 6.8G6 were deposited with the ATCC, ATCC accession numbers PTA-3649 and -3645, respectively.
Biogen: US7,465,449 - In some embodiments, the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by the hybridoma 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10, 6.2A1, 6.2E5 , 7.1G10, 7.7G5, or 7.1C5.
Centocor (J &J): US7,550,142; US7,163,681 For example, in US 7,550,142 - an antibody having human heavy chain and human light chain variable regions comprising the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.
Seattle Genetics: 15H3 (Ryan MC., Et al Cancer Res April 15, 2012; 72 (Supplement 8): 4630) (4D CEACAM5 (Molecule 5 cell adhesion related to carcinoembryonic antigen) Nucleotide Access of Genbank No. M17303 Genbank version No. M17303.1 GI: 178676 Date of GenBank registration update: June 23, 201008: 47 AM Polypeptide Access of Genbank No. AAB59513 Genbank version No. AAB59513.1 GI: 178677 Update date of GenBank registration: June 23, 201008: 47 AM Cross-references Beauchemin N., et al Mol. Cell. Biol. 7 (9), 3221-3230 (1987) Other information Official symbol: CEACAM5 Other Pseudonyms: CD66e, CEA Other Designations: 100 meconium antigen ANTIBODIES AstraZeneca-Medlmmune: US 20100330103; US20080057063; US20020142359 - for example, an antibody having complementarity determining regions (CDRs) with the following sequences: heavy chain; CDR1 - DNYMH, CDR2 - WIDPENGDTE YAPKFRG, CDR3 - LIYAGYLAMD Y; and light chain CDR1 SASSSVTYMH, CDR2 - STSNLAS, CDR3 - QQRSTYPLT.
- Hybridoma 806.077 deposited as deposit no. 96022936 of the European Collection of Cell Cultures (ECACC).
Research Corporation Technologies, Inc .: US5,04,507 Bayer Corporation: US6,013,772 BioAlliance: US7,982,017; US7,674,605 • US 7,674,605 an antibody comprising the heavy chain variable region sequence of the amino acid sequence of SEQ ID NO: 1, and the light chain variable region sequence of the amino acid sequence of SEQ ID NO: 2. an antibody comprising the heavy chain variable region sequence of the amino acid sequence of SEQ ID NO: 5, and the light chain variable region sequence of the amino acid sequence of SEQ ID NO: 6.
Celltech Therapeutics Limited: US5,877,293 The Dow Chemical Company: US5,472,693; US6,417,337; US6,333,405 US5,472,693 - for example, ATCC No. CRL-11215 US6,417,337 - for example, ATCC CRL-12208 US6,333,405 - for example, ATCC CRL-12208 Im unomedics, Inc .: US7,534,431; US7,230,084; US7,300,644; US6,730,300; US20110189085 an antibody having the CDRs of the light chain variable region comprises: CDR1 comprises KASQDVGTSVA (SEQ ID NO: 20); CDR2 comprises WTSTRHT (SEQ ID NO: 21); and CDR3 comprises QQYSLYRS (SEQ ID NO: 22); and the CDRs of the heavy chain variable region of said anti-CEA antibody comprise: CDR1 comprises TYWMS (SEQ ID NO: 23); CDR2 comprises E1HPDSSTINYAPSLKD (SEQ ID NO: 24); Y CDR3 comprises LYFGFPWFAY (SEQ ID NO: 25).
US20100221175; US20090092598; US20070202044; US20110064653; US20090185974; US20080069775. (42) MET (proto-oncogene met; hepatocyte growth factor receptor) Nucleotide Access of Genbank No. M35073 Genbank version No. M35073.1 GI: 187553 Date of GenBank registration update: March 6, 2012 5: 12 AM Polypeptide Access of Genbank No. AAA59589 Genbank version No. AAA59589.1 GI: 553531 Update date of GenBank registration: March 6, 201211: 12 AM Cross-references 15 Dean M., et al Nature 318 (6044), 385-388 (1985) Other information Official Symbol: MET Other Pseudonyms: AUTS9, HGFR, RCCP2, c-Met Other Designations: HGF receptor; HGF / SF receiver; SF receiver; hepatocyte growth factor receptor; tyrosine kinase of the proto-oncogene met; proto-oncogene c-Met; receiver dispersion factor; Met protein tyrosine kinase ANTIBODIES Abgenix / Pfizer: US20100040629 for example, the antibody produced by the hybridoma 13.3.2 having the accession number of the American Type Culture Collection (ATCC) PTA-5026; the antibody produced by hybridoma 9.1.2 having the accession number of ATCC PTA-5027; the antibody produced by the 8.70.2 hybridoma having accession number of ATCC PTA-5028; or the antibody produced by the hybridoma 6.90.3 having the accession number of ATCC PTA-5029.
Amgen / Pfizer: US20050054019 for example, an antibody comprising a heavy chain having the amino acid sequences set forth in SEQ ID NO: 2 wherein X2 is glutamate and X4 is serine and a light chain having the amino acid sequence set forth in SEQ ID NO: 4 in where X8 is alanine, without the signal sequences; an antibody that comprises a heavy chain having the amino acid sequences set forth in SEQ ID NO: 6 and a light chain having the amino acid sequence set forth in SEQ ID NO: 6 NO: 8, without the signal sequences; an antibody comprising a heavy chain having the amino acid sequences set forth in SEQ ID NO: 10 and a light chain having the amino acid sequence set forth in SEQ ID NO: 10; NO: 12, without the signal sequences; or an antibody comprising a heavy chain having the amino acid sequences set forth in SEQ ID NO: 14 and a light chain having the amino acid sequence set forth in SEQ ID NO: 14.
NO: 16, without the signal sequences.
Agouron Pharmaceuticals (Now Pfizer): US20060035907 Eli Lilly: ÜS20100129369 Genentech: US5,686,292; US20100028337; US20100016241; US20070129301; US20070098707; US20070092520, US20060270594; US20060134104; US20060035278; US20050233960; US20050037431 US 5,686,292 - for example, ATCC HB-11894 and ATCC HB-11895 US 20100016241 - for example, ATCC HB-11894 (hybridoma 1A3.3.13) or HB-11895 (hybridoma 5D5.11.6) National Defense Medical Center, Taiwan: Lu RM., Et al Biomaterials.2011 Apr; 32 (12): 3265-74.
Novartis: US20090175860 - for example, an antibody comprising the CDR1, CDR2, and CDR3 sequences of the 4687 heavy chain, wherein the CDR1, CDR2, and CDR3 sequences of the 4687 heavy chain are residues 26-35, 50-65, and 98-102, respectively, of SEQ ID NO: 58; and the CDR1, CDR2, and CDR3 sequences of the 5097 light chain, wherein the CDR1, CDR2, and CDR3 sequences of the 5097 light chain are residues 24-39, 55-61, and 94-100 of SEQ. DO NOT: 37 Pharmacia Corporation: US20040166544 Pierre Fabre: US20110239316, US20110097262, US20100115639 Sumsung: US 20110129481 - for example, a monoclonal antibody produced from a hybridoma cell having the accession number KCLRF-BP-00219 or the accession number of KCLRF-BP-00223.
Samsung: US 20110104176 - for example, an antibody produced by a hybridoma cell having the accession number: KCLRF-BP-00220.
Medical School of the University of Turin: DN-30 Pacchiana G., et al J Biol Chem. 2010 Nov 12; 285 (46): 36149-57 Van Andel Research Institute: Jiao Y., et al Mol Biotechnol. 2005 Sep; 31 (1): 41-54. (43) MUC1 (Mucin 1, associated with the cell surface) Nucleotide Access of Genbank No. J05581 Genbank version No. J05581.1 GI: 188869 Date of GenBank registration update: June 23, 201008: 48 AM Polypeptide Access of Genbank No. AAA59876 Genbank version No. AAA59876.1 GI: 188870 Update date of the GenBank registration: June 23, 201008: 48 AM Cross-references Gendler S.J., et al J. Biol. Chem. 265 (25), 15286-15293 (1990) Other information Official Symbol: MUC1 Other Pseudonyms: RP11-263K19.2, CD227, EMA, H23AG, KL-6, MAM6, MUC-1, MUC-l / SEC, MUC-l / X, MUC1 / ZD, PEM, PEMT, PUM Other Designations: DF3 antigen; H23 antigen; DF3 antigen associated with breast carcinoma; mucin associated with carcinoma; episialin; krebs von den Lungen-6; mucin 1, transmembrane; mucin-1; reactive urinary mucin with peanut; polymorphic epithelial mucin; epithelial mucin associated with tumor; antigen of the epithelial membrane associated with tumor; mucin associated with tumor ANTIBODIES AltaRex- Quest Pharma Tech: US 6,716,966 - for example, an antibody for Alt-1 produced by the ATCC hybridoma No. PTA-975.
AltaRex- Quest Pharma Tech: US7,147,850 CRT: 5E5 - Sorensen AL., Et al Glycobiology vol. 16 no. 2 p.p. 96-107, 2006; HMFG2 - Burchell J., et al Cancer Res. , 47, 5476-5482 (1987) Glycotope GT-MAB: GT-MAB 2.5-GEX (Website: http://www.glycotope.com/pipeline/pankomab-gex) Immunogen: US7,202,346 - for example, MJ-170 antibody: hybridoma cell line MJ-170 no. of ATCC access PTA-5286; monoclonal antibody MJ-171: hybridoma cell line MJ-171 no. of access of ATCC PTA-5287; monoclonal antibody MJ-172: hybridoma cell line MJ-172 no. of access of ATCC PTA-5288; or monoclonal antibody MJ-173: hybridoma cell line MJ-173 no. Access Code PTA-5302 Immunomedics: US 6,653,104 Ramot Tel Aviv Uni: US7,897,351 Regents Uní. CA: US 7,183,388; US20040005647; US20030077676.
Roche GlycArt: US8,021,856 Russian National Center for Oncological Research: Imuteran-Ivanov PK., Et al Biotechnol J. 2007 Jul; 2 (7): 863-70 Technical University of Braunschweig: (IIB6, HT186-B7, HT186-D11, HT186-G2, HT200-3A-C1, HT220-M-D1, HT220-M-G8) - Thie H., et al. PLoS One. 2011 Jan 14; 6 (1): el5921 (44) CA9 (Carbonic anhydrase IX) Nucleotide Access of Genbank No. X66839 Genbank version No. X66839.1 GI: 1000701 Update date of GenBank registration: February 2, 201110: 15 AM Polypeptide Access of Genbank No. CAA47315 Genbank version No. CAA47315.1 GI: 1000702 Date of GenBank registration update: February 2, 201110: 15 AM Cross-references Pastorek J., et al Oncogene 9 (10), 2877-2888 (1994) Other information Official Symbol: CA9 Other Pseudonyms: CAIX, MN Other Designations: CA-IX; P54 / 58N; antigen G250 associated with RCC; G250 protein associated with RCC; carbonate dehydratase IX; carbonic anhydrase 9; carbonic dehydratase; membrane antigen MN; pMWl; G250 antigen associated with renal cell carcinoma ANTIBODIES Abgenix / Amgen: US20040018198 Affibody: Molecules of Affibody anti-CAIX (http://www.affibody.com/en/Product-Portfolio/Pipeline/) Bayer: US7,462,696 Bayer / Morfosys: mAb 3ee9 - Petrul HM., Et al Mol Cancer Ther. 2012 Feb; 11 (2): 340-9 Harvard Medical School: Antibodies G10, G36, G37, G39, G45, G57, G106, G119, G6, G27, G40 and G125. Xu C., et al PLoS One. 2010 Mar 10; 5 (3): e9625 Institute of Virology, Slovak Academy of Sciences (Bayer) - US5,955,075 - for example, M75 - Access No. of ATCC HB 11128 or MN12 - Accession No. of ATCC HB 11647 Institute of Virology, Slovak Academy of Science: US7,816,493 - for example, monoclonal antibody M75 which is secreted from hybridoma VU-M75, which was deposited in the American Type Culture Collection under No. ATCC HB 11128; or monoclonal antibody V / 10 hybridoma secreted V / 10- VU, which was deposited with International Depositary Authority Belgian Coordinated Collection of Microorganisms (BCCM) Laboratorium voor Moleculaire in Bioloqie-Plasmidencollectie (LMBP) in Gent Universeit Ghent, Belgium, under accession number LMBP 6009CB.
Institute of Virology, Slovak Academy of Sciences - US20080177046; US20080176310; US20080176258; US20050031623 Novartis: US20090252738 Wilex: US7,691,375 - for example, the antibody produced by the hybridoma cell line DSM ASC 2526.
Wilex: US20110123537; Rencarex: Kennett RH., Et al Curr Opin Mol Ther. 2003 Feb; 5 (1): 70-5 Xencor: US20090162382 (45) EGFRvIII (Epidermal growth factor receptor (EGFR), transcript variant 3, Nucleotide Access of Genbank No. NM 201283 Genbank version No. NM 201283.1 GI: 41327733 Update date of GenBank registration: September 30, 201201: 47 PM Polypeptide Access of Genbank No. NP_958440 Genbank version No. NP_958440.1 GI: 41327734 Date of GenBank registration update: September 30, 201201: 47 PM Cross-references Batra SK., Et al Cell Growth Differ 1995; 6: 1251-1259.
ANTIBODIES: US7,628,986 and US7,736,644 (Amgen) For example, an amino acid sequence of the heavy chain variable region selected from the group consisting of SEQ ID NO: 142 and variants and an amino acid sequence of the light chain variable region selected from the group consisting of: SEQ ID NO: 144 and variants.
US20100111979 (Amgen) For example, an antibody comprising a heavy chain amino acid sequence comprising: CDR1 consisting of a sequence selected from the group consisting of the amino acid sequences for the CDR1 region of antibodies 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4) , 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEO ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO : 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17); CDR2 consisting of a sequence selected from the group consisting of the amino acid sequences for the CDR2 region of antibodies 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4) , 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 10) NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17); Y CDR3 consisting of a sequence selected from the group consisting of the amino acid sequences for the CDR3 region of antibodies 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4) , 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 10) NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16) and 333 (SEQ ID NO: 17).
US20090240038 (Amgen) For example, an antibody having at least one of the heavy or light chain polypeptides comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO: 142, SEQ ID NO: 144, and any combination thereof.
US20090175887 (Amgen) For example, an antibody having a heavy chain amino acid sequence selected from the group consisting of the heavy chain amino acid sequence of antibody 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16). and 333 (SEQ ID NO: 17).
US20090156790 (Amgen) For example, an antibody having the heavy chain polypeptide and a light chain polypeptide, wherein at least one of the heavy or light chain polypeptides comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO: 142, SEQ ID NO: 144, and any combination thereof.
US20090155282, US20050059087 and US20050053608 (Amgen) For example, a heavy chain amino acid sequence of the antibody selected from the group consisting of the heavy chain amino acid sequence of antibody 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ. ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16) and 333 (SEQ ID NO: 17).
MR1-1 (US7,129,332; Duke) For example, a variant antibody having the sequence of SEQ ID NO. 18 with substitutions S98P-T99Y in CDR3 VH and F92W in CDR3 VL.
L8A4, H10, Y10 (Wikstrand CJ., Et al Cancer Res. 1995 Jul 15; 55 (14): 3140-8; Duke) US20090311803 (Harvard University) For example, SEQ ID NO: 9 for the heavy chain variable region of the antibody, and SEQ ID NO: 3 for the amino acid sequences of the light chain variable region US20070274991 (EMD72000, also known as matuzumab, Harvard University) For example, SEQ ID NOs: 3 and 9 for light chain and heavy chain, respectively US6,129,915 (Schering) For example, SEQ ID NOs: 1, 2, 3, 4, 5 and 6. mAb CH12 - Wang H., et al FASEB J. 2012 Jan; 26 (1): 73-80 (Shanghai Cancer Institute).
RAbDMvlll - Gupta P., et al BMC Bíotechnol. 2010 Oct 7; 10: 72 (Stanford University Medical Center). mAb Ua30 - Ohman L., et al Tumor Biol.2002 Mar- Apr; 23 (2): 61-9 (University of Uppsala).
Han DG., Et al Nan Fang Yi Ke Da Xue Xue Bao. 2010 Jan; 30 (1): 25-9 (University of Xi'an Jiaotong). (46) CD33 (Molecula CD33) Nucleotide Access of Genbank No. M_23197 Genbank version No. NM_23197.1 GI: 180097 Update date of GenBank registration: June 23, 201008: 47 AM Polypeptide Access of Genbank No. AAA51948 Genbank version No. AAA51948.1 GI: 188098 Update date of GenBank registration: June 23, 201008: 47 AM Cross-references Siramons D. , et al J. Immunol. 141 (8), 2797-2800 (1988) Other information Official Symbol: CD33 Other Pseudonyms: SIGLEC-3, SIGLEC3, p67 Other Designations: antigen CD33 (gp67); gp67; CD33 surface antigen of myeloid cells; lectin 3 similar to Ig binding to sialic acid; lectin similar to Ig binding to sialic acid ANTIBODIES H195 (Lintuzumab) - Race A., et al Leuk Lymphoma. 2009 Aug; 50 (8): 1336-44; US6,759,045 (Seattle Genetics / Immunomedics) mAb OKT9: Sutherland, D.R. et al. Proc Nati Acad Sci USA 78 (7): 4515-4519-1981, Schneider.C., Et al J Biol Chem 257, 8516-8522 (1982) mAb E6: Hoogenboom, H.R., et al. J Immunol 144, 3211-3217 (1990) US6,590,088 (Human Genome Sciences) For example, SEQ ID NOs: 1 and 2 and no. access ATCC 97521 US7,557,189 (Imunogen) For example, an antibody or fragment thereof comprising a heavy chain variable region comprising three CDRs having the amino acid sequences of SEQ ID NOs: 1-3 and a light chain variable region comprising three CDRs having the sequences of amino acids of SEQ ID NOs: 4-6. (47) CD19 (Molecula CD19) Nucleotide Access of Genbank No. NM_001178098 Genbank version No. NM_001178098.1 GI: 296010920 Date of GenBank registration update: September 10, 201212: 43 AM Polypeptide Access of Genbank No. NP_001171569 Genbank version No. NP_001171569.1 GI: 296010921 Date of GenBank registration update: September 10, 201212: 43 AM Cross-references Tedder TF., Et al J. Immunol. 143 (2): 712-7 (1989) Other information Official Symbol: CD19 Other Pseudonyms: B4, CVID3 Other Designations: CD19 antigen of B lymphocytes; B4 surface antigen of B lymphocytes; Leu-12 T cell surface antigen; CD19 antigen differentiation ANTIBODIES Immunogen: HuB4 - Al-Katib AM., Et al Clin Cancer Res. 2009 Jun 15; 15 (12): 4038-45. 4G7: Kügler M., et al Protein Eng Des Sel. 2009 Mar; 22 (3): 135-47 For example, the sequences in Fig. 3 of Knappik, A. et al J Mol Biol 2000 Feb; 296 (l): 57-86 AstraZeneca / Medlmmune: MEDI-551 - Herbst R., et al J Pharmacol Exp Ther. 2010 Oct; 335 (1): 213-22 Glenmark Pharmaceuticals: GBR-401 Hou S., et al Mol Cancer Ther November 2011 10 (Abstract Congress Supplement) C164 US7,109,304 (Immunomedics) For example, an antibody comprising the sequence of hAl9Vk (SEQ ID NO: 7) and the sequence of hA19VH (SEQ ID NO: 10) US7,902,338 (Immunomedics) For example, an antibody or antigen-binding fragment thereof comprising the CDR sequences of the CDR1 light chain complementarity determining region of SEQ ID NO: 16 (KASQSVDYDGDSYLN); CDR2 of SEQ ID NO: 17 (DASNLVS); and CDR3 of SEQ ID NO: 18 (QQSTEDPWT) and CDR1 heavy chain CDR sequences of SEQ ID NO: 19 (SYWMN); CDR2 of SEQ ID NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3 of SEQ ID NO: 21 (RETTTVGRYYYAMDY) and also comprises sequences of the structural region (FR) and human antibody constant region with one or more amino acid residues of the substituted structural region from the corresponding structural region sequences of the parental murine antibody, and wherein said substituted FR residues comprise the substitution of serine by phenylalanine in the Kabat residue 91 of the heavy chain variable region.
Medarex: MDX-1342 - Cardarelli PM., Et al Cancer Immunol Immunother. 2010 Feb; 59 (2): 257-65.
MorphoSys / Xencor: MOR-208 / XmAb-5574 - Zalevsky J., et al Blood. 2009 Apr 16; 113 (16): 3735-43 US7,968,687 (Seattle Genetics) An antibody or antigen binding fragment comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 24. 4G7 chim - Lang P., et al Blood. 2004 May 1; 103 (10): 3982-5 (University of Tübingen) For example, Fig. 6 and SEQ ID NO: 80 of US20120082664 Zhejiang University School of Medicine: 2E8 - Zhang J., et al J Drug Target. 2010 Nov; 18 (9): 675-8 (48) IL2RA (Interleukin 2 receptor, alpha); NCBI Reference Sequence: NM_000417.2); Nucleotide Access of Genbank No. NM_000417 Genbank version No. NM_000417.2 GI: 269973860 Update date of GenBank registration: September 9, 201204: 59 PM Polypeptide Access of Genbank No. NP 000408 Genbank version No. NP_000408.1 GI: 4557667 Update date of GenBank registration: September 9, 201204: 59 PM Cross-references Kuziel W.A., et al J. Invest. Dermatol. 94 (SUPL 6), 27S-32S (1990) Other information Official Symbol: IL2RA Other Pseudonyms: RP11-536K7.1, CD25, IDDM10, IL2R, TCGFR Other Designations: alpha subunit of the FIL-2 receptor; IL-2-RA; alpha subunit of IL-2R; IL2-RA; TAC antigen; alpha subunit of the interleukin-2 receptor; P55 ANTIBODIES US6,383,487 (Novartis / UCL: Baxilisimab [Simulect]) US6,521,230 (Novartis / UCL: Baxilisimab [Simulect]) For example, an antibody having an antigen binding site comprises at least one domain comprising CDR1 having the amino acid sequence in SEQ ID NO: 7, CDR2 having the amino acid sequence in SEQ ID NO: 8, and CDR3 that has the amino acid sequence in SEQ ID NO: 9; or said CDR1, CDR2 and CDR3 taken in The sequence as a whole comprises an amino acid sequence that is at least 90% identical to SEQ ID NOs: 7, 8 and 9 taken in sequence as a whole.
Daclizumab - Rech AJ., Et al Ann N and Acad Sci. 2009 Sep; 1174: 99-106 (Roche) (49) AXL (tyrosine kinase of the AXL receptor) Nucleotide Access of Genbank No. M76125 Genbank version No. M76125.1 GI: 292869 Date of GenBank registration update: June 23, 201008: 53 AM Polypeptide Access of Genbank No. AAA61243 Genbank version No. AAA61243.1 GI: 29870 Update date of the GenBank registry: June 23, 201008: 53 AM Cross-references O'Bryan J.P., et al Mol. Cell. Biol. 11 (10), 5016-5031 (1991); Bergsagel P.L., et al J. Immunol. 148 (2), 590-596 (1992) Other information Official Symbol: AXL Other Pseudonyms: JTK11, UFO Other Designations: AXL oncogene; sequence / AXL transformant gene; AXL oncogene; UFO protein receptor tyrosine kinase ANTIBODIES YW327.6S2 - Ye X., et al Oncogene. 2010 Sep 23/29 (38): 5254-64. (Genentech) BergenBio: BGB324 (http://www.bergenbio.com/BGB324) (50) CD30 - TNFRSF8 (Superfamily of tumor necrosis factor receptors, member 8) Nucleotide Access of Genbank No. M83554 Genbank version No. M83554.1 GI: 180095 Date of GenBank registration update: June 23, 201008: 53 AM Polypeptide Access of Genbank No. AAA51947 Genbank version No. AAA51947.1 GI: 180096 Update date of GenBank registration: June 23, 201008: 53 AM Cross-references Durkop H., et al Cell 68 (3), 421-427 (1992) Other information Official Symbol: TNFRSF8 Other Pseudonyms: CD30, D1S166E, Ki-1 Other Designations: CD30L receiver; antigen Ki-1; cytokine CD30 receptor; CD30 antigen for lymphocyte activation; superfamily of receptors tumor necrosis factor, member 8 (5D BCMA (B cell maturation antigen) - TNFRSF1 7 (Tumor necrosis factor receptor superfamily, member 17) Nucleotide Access of Genbank No. Z29574 Genbank version No. Z29574.1 GI: 471244 Date of GenBank registration update: February 2, 201110: 40 AM Polypeptide Access of Genbank No. CAA82690 Genbank version No. CAA82690.1 GI: 471245 Update date of GenBank registration: February 2, 201110: 40 AM Cross-references Laabi Y. et al Nucleic Acids Res. 22 (7), 1147-1154 (1994) Other information Official Symbol: TNFRSF17 Other Pseudonyms: BCM, BCMA, CD269 Other Designations: B cell maturation antigen; B cell maturation factor; B cell maturation protein; Tumor necrosis factor receptor superfamily, member 17 (52) Ags of CT - CTA (Testicle antigens of Cancer) Cross-references Fratta E. , et al Mol Oncol. 2011 Apr; 5 (2): 164-82; Lira SH. , et al Ara J Blood Res. 2012; 2 (1): 29-35. (53) CD174 (Lewis Y) - FUT3 (fucosyltransferase 3 (galactoside 3 (4) -L-fucosyltransferase Lewis blood group) Nucleotide Access of Genbank No. NM00019 Genbank version No. NM000149.3 GI: 148277008 GenBank registration update date: June 26, 201204: 49 PM Polypeptide Access of Genbank No. NP_000140 Genbank version No. NP_000140.1 GI: 4503809 Update date of GenBank registration: June 26, 201204: 49 PM Cross-references Kukowska-Latallo, J.F., et al Genes Dev. 4 (8), 1288-1303 (1990) Other information Official Symbol: FUT3 Other Pseudonyms: CD174, FT3B, FucT-III, LE, Les Other Designations: Lewis FT; alpha- (1,3 / 1,4) -fucosyltransferase; blood group Lewis alpha-4- fucosyltransferase; fucosyltransferase III; galactoside 3 (4) -L-fucosyltransferase (54) CLEC14A (family 14 of the C-type lectin domain, member A; Genbank Access No. NM1 75060) Nucleotide Access of Genbank No. NM175060 Genbank version No. NM175060.2 GI: 371123930 Update date of GenBank registration: April 1, 201203: 34 PM Polypeptide Access of Genbank No. NP_778230 Genbank version No. NP_778230.1 GI: 28269707 Update date of GenBank registration: April 1, 201203: 34 PM Other information Official Symbol: CLEC14A Other Pseudonyms: UNQ236 / PR0269, C14orf27, CEG1, EGFR-5 Other Designations: family 14 of type C lectin domain, member A; protein containing domain similar to CIECT and EGF; 5 receptor of the epidermal growth factor (55) GRP78 - HSPA5 (protein 70 kDa thermal shock (protein regulated by glucose, 78 kDa) Nucleotide Access of Genbank No. NM005347 Genbank version No. NM005347.4 GI: 305855105 GenBank registration update date: September 30, 201201: 42 PM Polypeptide Access of Genbank No. NP_005338 Genbank version No. NP_005338.1 GI: 16507237 Date of GenBank registration update: September 30, 201201: 42 PM Cross-references Ting J., et al DNA 7 (4), 275-286 (1988) Other information Official Symbol: HSPA5 Other Pseudonyms: BIP, GRP78, MIF2 Other Designations: 78 kDa protein regulated by glucose; binding protein grp78 to Ca (2+) lumen of the endoplasmic reticulum; immunoglobulin heavy chain binding protein (56) CD70 (Molecule CD70) L08096 Nucleotide Access of Genbank No. L08096 Genbank version No. L08096.1 GI: 307127 Update date of the GenBank registry: June 23, 201208: 54 AM Polypeptide Access of Genbank No. AAA36175 Genbank version No. AAA36175.1 GI: 307128 Update date of GenBank registration: June 23, 201208: 54 AM Cross-references Goodwin R.G., et al Cell 73 (3), 447-456 (1993) Other information Official Symbol: CD70 Other Pseudonyms: CD27L, CD27LG, TNFSF7 Other designations: ligand CD27; CD27-L; CD70 antigen; Ki-24 antigen; CD70 surface antigen; superfamily of tumor necrosis factor (ligand), member 7; Tumor necrosis factor ligand superfamily, member 7 ANTIBODIES MDX-1411 against CD70 (Medarex) hlF6 (Oflazoglu, E., et al, Clin Cancer Res.2008 Oct 1; 14 (19): 6171-80; Seattle Genetics) For example, see US20060083736 SEQ ID NOs: 1, 2, 11 and 12 and Fig.1. (57) Stem cell-specific antigens. For example : • 5T4 (see entry (63) below) CD25 (see entry (48) above) CD32 or Polypeptide Access of Genbank No. ABK42161 Genbank version No. ABK42161.1 GI: 117616286 Update date of the GenBank registry: July 25, 2007 03:00 PM LGR5 / GPR49 or Nucleotide Genbank Access No. NM_003667 Genbank Version No. NM_003667.2 GI: 24475886 Update date of GenBank registration: July 22, 201203: 38 PM or Polypeptide Access of Genbank No. NP_003658 Genbank version No. NP 003658.1 GI: 4504379 Update date of GenBank registration: July 22, 201203: 38 PM Prominin / CD133 Nucleotide Access of Genbank No. NM 006017 Genbank version No. NM_006017.2 GI: 224994187 Update date of GenBank registration: September 30, 2012 01:47 PM or Polypeptide Access of Genbank No. NP 006008 Genbank version No. NP_006008.1 GI: 5174387 Update date of GenBank registration: September 30, 2012 01:47 PM (58) ASG-5 Cross-references (Smith L.M., et al AACR 2010 Annual Meeting (abstract # 2590); Gudas J.M., et al AACR 2010 Annual Meeting (abstract # 4393) ANTIBODIES Anti-AGS-5 antibody: M6.131 (Smith, L.M., et al. AACR 2010 Annual Meeting (abstract # 2590) (59) ENPP3 (Pyro fuction of eytonucleotide / f-eesterase 3) Nucleotide Access of Genbank No. AF005632 Genbank version No. AF005632.2 GI: 4432589 Update date of GenBank registration: March 10, 201009: 41 PM Polypeptide Access of Genbank No. AAC51813 Genbank version No. AAC51813.1 GI: 2465540 Date of GenBank registration update: March 10, 201009: 41 PM Cross-references Jin-Hua P., et al Genomics 45 (2), 412-415 (1997) Other information Official Symbol: ENPP3 Other Pseudonyms: RP5-988G15.3, B10, CD203c, NPP3, PD-IBETA, PDNP3 Other Designations: E-NPP 3; dJ1005H11.3 (phosphodiesterase I / nucleotide pyrophosphatase 3); dJ914N13.3 (phosphodiesterase I / nucleotide pyrophosphatase 3); member 3 of the ectonucleotide pyrophosphatase / phosphodiesterase family; gpl30RB13-6; Phosphodiesterase I beta; phosphodiesterase I / nucleotide pyrophosphatase 3; phosphodiesterase-I beta (60) PRR4 (4 rich in proline (lacrimal)) Nucleotide Access of Genbank No. NM 007244 Genbank version No. NM 007244.2 GI: 154448885 Update date of GenBank registration: June 28, 201212: 39 PM Polypeptide Access of Genbank No. NP_009175 Genbank version No. NP_009175.2 GI: 154448886 Date of GenBank registration update: June 28, 201212: 39 PM Cross-references Dickinson D.P., et al Invest. Ophthalmol. Vis. Sci. 36 (10), 2020-2031 (1995) Other information Official Symbol: PRR4 Other Pseudonyms: LPRP, PROL4 Other Designations: protein rich in lacrimal proline; proline-rich protein 4 associated with nasopharyngeal carcinoma; proline-rich polypeptide 4; protein 4 rich in proline (6D GCC - GUCY2C (cyclasic guanylate 2C (heat-stable enterotoxin receptor) Nucleotide Access of Genbank No. NM__004963 Genbank version No. NM_004963.3 GI: 222080082 Update date of GenBank registration: September 2, 201201: 50 PM Polypeptide Access of Genbank No. NP_004954 Genbank version No. NP_004954.2 GI: 222080083 Date of GenBank registration update: September 2, 201201: 50 PM Cross-references De Sauvage F.J., et al J. Biol. Chem. 266 (27), 17912-17918 (1991); Singh S., et al Biochem. Biophys. Beef.
Commun. 179 (3), 1455-1463 (1991) Other information Official Symbol: GUCY2C Other Pseudonyms: DIAR6, GUC2C, MUCIL, STAR Other Designations: GC-C; STA receiver; guanilil cielasa C; hSTAR; heat-stable enterotoxin receptor; intestinal guanylate cyclase (62) Liv-1 - SLC39A6 (family 39 of transporters and solutes (zinc transporter), member 6) Nucleotide Access of Genbank No. U41060 Genbank version No. U41060.2 GI: 12711792 Update date of GenBank registration: November 30, 200904: 35 PM Polypeptide Access of Genbank No. AAA96258 Genbank version No. ADA96258.2 GI: 12711793 Update date of GenBank registration: November 30, 200904: 35 PM Cross-references Taylor KM., Et al Biochim Biophys Acta. 2003 Apr 1; 1611 (1-2): 16-30 Other information Official Symbol: SLC39A6 Other Pseudonyms: LIV-1 Other Designations: LIV-1 protein, regulated by estrogen; ZIP-6; LIV-1 protein regulated by estrogen; family 39 of transporters and solutes (metal ion transporter), member 6; family of transporters and solutes 39 member 6; ZIP6 zinc conveyor; protein 6 similar to zrt- and Irt (63) 5T4, Trophoblast glycoprotein, TPBG - TPBG (trophoblast glycoprotein) Nucleotide Access of Genbank No. AJ012159 Genbank version No. AJ012159.1 GI: 3805946 Date of GenBank registration update: February 1, 201110: 27 AM Polypeptide Access of Genbank No. CAA09930 Genbank version No. CAA09930.1 GI: 3805947 Date of GenBank registration update: 1 February 201110: 27 AM Cross-references King K.W., et al Biochim. Biophys. Acta 1445 (3), 257-270 (1999) Other information • Official Symbol: TPBG Other Pseudonyms: 5T4, 5T4AG, M6P1 • Other Designations: 5T4 oncofetal antigen; oncofetal trophoblast glycoprotein 5T4; 5T4 oncotrophoblast glycoprotein (64) CD56 - NCMA1 (Molecule 1 of adhesion to neural cells) Nucleotide Access of Genbank No. NM_000615 Genbank version No. NM_000615.6 GI: 336285433 Date of GenBank registration update: September 23, 201202: 32 PM Polypeptide Access of Genbank No. NP_000606 Genbank version No. NP_000606.3 GI: 94420689 Date of GenBank registration update: September 23, 201202: 32 PM Cross-references Dickson, G., et al, Cell 50 (7), 1119-1130 (1987) Other information Official Symbol: NCAM1 Other Pseudonyms: CD56, MSK39, NCAM Other Designations: antigen recognized by the monoclonal antibody 5.1H11; Neural cell adhesion molecule, NCAM ANTIBODIES Immunogen: HuN901 (Smith SV., Et al Curr Opin Mol Ther, 2005 Aug; 7 (4): 394-401) For example, see humanized murine N901 antibody. See Fig. Ib and that of Roguska, M.A., et al Proc Nati Acad Sci USA Feb 1994; 91: 969-973. (65) CanAg (Antigen associated with tumor CA242) Cross-references Haglund C., et al Br J Cancer 60: 845-851, 1989; Baeckstrom D., et al J Biol Chem 266: 21537-21547, 1991 ANTIBODIES huC242 (Tolcher AW et al., J Clin Oncol., 2003 Jan 15; 21 (2): 211-22; Immunogen) For example, see US20080138898A1 SEO ID NO: 1 and 2 (66) FOLR1 (Receiver 1 of Fola to) Nucleotide Access of Genbank No. J05013 Genbank version No. J05013.1 GI: 182417 Update date of the GenBank registry: June 23, 201008: 47 AM Polypeptide Access of Genbank No. AAA35823 Genbank version No. AAA35823.1 GI: 182418 Update date of GenBank registration: June 23, 201008: 47 AM Cross-references Elwood P.C., et al J. Biol. Chem. 264 (25), 14893-14901 (1989) Other information Official Symbol: FOLR1 Other Pseudonyms: FBP, FOLR Other Designations: FR-alpha; KB FBP cells; adult folate binding protein; folate binding protein; alpha folate receptor; folate receptor, adult; MOvl8 antigen associated with ovarian tumor ANTIBODIES M9346A - Whiteman KR., Et al Cancer Res April 15, 2012; 72 (Supplement 8): 4628 (Immunogen) (67) GPNMB (Glycoprotein (transmembrane) nmb) Nucleotide Access of Genbank No. X76534 Genbank version No. X76534.1 GI: 666042 Update date of GenBank registration: February 2, 201110: 10 AM Polypeptide Access of Genbank No. CAA54044 Genbank version No. CAA54044.1 GI: 666043 Update date of GenBank registration: February 2, 201110: 10 AM Cross-references Weterman M .A. , et al Int. J. Cancer 60 (1), 73-81 (nineteen ninety five) Other information Official Symbol: GPNMB Other Pseudonyms: UNQ1725 / PR09925, HGFIN, NMB Other Designations: NMB glycoprotein; protein similar to glycoprotein nmb; osteoactivin; transmembrane glycoprotein HGFIN; NMB transmembrane glycoprotein ANTIBODIES Celldex Therapeutics: CR011 (Tse KF., Et al Clin Cancer Res. 2006 Feb 15; 12 (4): 1373-82) For example, see EP1827492B1 SEQ ID NO: 22, 24, 26, 31, 33 and 35 (68) TIM-1 - HAVCR1 (Cell Receptor 1 of the virus of hepa ti tis A) Nucleotide Access of Genbank No. AF043724 Genbank version No. AF043724.1 GI: 2827453 Update date of the GenBank registry: March 10, 201006: 24 PM Polypeptide Access of Genbank No. AAC39862 Genbank version No. AAC39862.1 GI: 2827454 Date of GenBank registration update: March 10, 201006: 24 PM Cross-references Feigelstock D., et al J. Virol. 72 (8), 6621-6628 (1998) Other information Official Symbol: HAVCR1 Other Pseudonyms: HAVCR, HAVCR-1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1, TIMD1 Other Designations: immunoglobulin domain protein 1 and mucin domain of T cells; protein 1 of the T cell membrane; molecule 1 of kidney damage (69) RG-l / Míndina tumor target of the prostate -Mindin / RG-1 Cross-references Parry R., et al Cancer Res. 2005 Sep 15; 65 (18): 8397-405 (70) B7-H4 - VTCN1 (Inhibitor 1 of activation of T cells containing the domain of set V) Nucleotide Access of Genbank No. BX648021 F Genbank version No. BX648021.1 GI: 34367180 Update date of GenBank registration: February 2, 201108: 40 AM Cross-references Sica GL., Et al Immunity. 2003 Jun; 18 (6): 849-61 Other information Official Symbol: VTCN1 Other Pseudonyms: RP11-229A19.4, B7-H4, B7H4, B7S1, B7X, B7h.5, PR01291, VCTN1 Other Designations: member of the B7 family, H4; member 1 of the superfamily B7; co-stimulatory B7x molecule of T cells; T7 cell B7x costimulatory molecule; inhibitor 1 of the activation of T cells containing the domain of set V; immuno-stimulating B7-H4 protein (7D PTK7 (Protein tyrosine kinase 7 PTK7) Nucleotide Access of Genbank No. AF447176 Genbank version No. AF447176.1 GI: 17432420 Date of GenBank registration update: November 28, 200801: 51 PM Polypeptide Access of Genbank No. AAL39062 Genbank version No. AAL39062.1 GI: 17432421 Update date of the GenBank registry: November 28, 200801: 51 PM Cross-references Park S.K., et al J. Blochem. 119 (2), 235-239 (nineteen ninety six) Other information Official Symbol: PTK7 Other Pseudonyms: CCK-4, CCK4 Other Designations: colon carcinoma kinase 4; inactive protein tyrosine kinase 7; pseudotyrosine kinase receptor 7; 7 similar to protein tyrosine kinase (72) CD37 (Molecula CD37) Nucleotide Access of Genbank No. NM_001040031 Genbank version No. NM_001040031.1 GI: 91807109 GenBank registration update date: July 29, 201202: 08 PM Polypeptide Access of Genbank No. NP_001035120 Genbank version No. NP_001035120.1 GI: 91807110 Date of GenBank registration update: July 29, 201202: 08 PM Cross-references Schwartz-Albiez R., et al J. Immunol. 140 (3), 905-914 (1988) Other information Official Symbol: CD37 Other Pseudonyms: GP52-40, TSPAN26 Other Designations: CD37 antigen; cell differentiation antigen 37; CD37 antigen of leukocytes; CD37 leukocyte surface antigen; tetraspanin-26; tspan-26 ANTIBODIES Boehringer Ingelheim: mAb 37.1 (Heider KH., Et al Blood, 2011 Oct 13; 118 (15): 4159-68) Trubion: CD37-SMIP (G28-1 scFv-Ig) (Zhao X. et al Blood, 2007; 110: 2569-2577) For example, see US20110171208A1 SEQ ID NO: 253 Immunogen: K7153A (Deckert J. et al Res Cancer April 15, 2012; 72 (Supplement 8): 4625) (73) CD138 - SDC1 (syndecan D Nucleotide Access of Genbank No. AJ551176 Genbank version No. AJ551176.1 GI: 29243141 Date of GenBank registration update: February 1, 201112: 09 PM Polypeptide Access of Genbank No. CAD80245 Genbank version No. CAD80245.1 GI: 29243142 Update date of GenBank registration: February 1, 201112: 09 PM Cross-references O'Connell FP., Et al Am J Clin Pathol. 2004 Feb; 121 (2): 254-63 Other information Official Symbol: SDC1 Other Pseudonyms: CD138, SDC, SYND1, syndecan Other Designations: antigen CD138; heparan sulfate proteoglycan fibroblast growth factor receptor; syndecan proteoglycan 1; sindecano-1 ANTIBODIES Biotest: Chimerized MAb (nBT062) - (Jagannath S., et al Poster ASH # 3060, 2010; Patent Application WIPO WO / 2010/128087) For example, see US20090232810 SEQ ID NO: 1 and 2 Immunogen: B-B4 (Tassone P., et al Blood 104_3688-3696) For example, see US20090175863A1 SEQ ID NO: 1 and 2 (74) CD74 (Molecule CD74, major complex of histocompatibility chain class ID invariant Nucleotide Access of Genbank No. NM 004355 Genbank version No. NM 004355.1 GI: 343403784 Update date of the GenBank registry: September 23, 201202: 30 PM Polypeptide Access of Genbank No. NP_004346 Genbank version No. NP_004346.1 GI: 10835071 Date of GenBank registration update: September 23, 201202: 30 PM Cross-references Kudo, J. et al Nucleic ñcids Res. 13 (24), 8827-8841 (1985) Other information Official Symbol: CD74 Other Pseudonyms: DHLAG, HLADG, II, Ia-GAMMA Other Designations: antigen CD74 (invariant polypeptide of the major histocompatibility complex, associated with the class II antigen); gamma chain of HLA class II histocompatibility antigen; invariant chain associated with HLA-DR antigens; HLA-DR-gamma; invariant chain associated with the; MHC HLA-DR gamma chain; Gamma chain of class II antigens; p33 ANTIBODIES Immunomedics: hLLl (Milatuzumab,) - Berkova Z. et al Expert Opin Investig Drugs. 2010 Jan; 19 (1): 141-9) For example, see US20040115193 SEQ ID NOs: 19, 20, 21, 22, 23 and 24 Genmab: HuMax-CD74 (see website) (75) Claudinas 5 - CLs (Claudinas) Cross-references Offner S., et al Cancer Immunol Immunother. 2005 May; 5 (5): 431-45, Suzuki H., et al Ann N and Acad Sci. 2012 Jul; 1258: 65-70) In humans, 24 members of the family have been described - see bibliographic reference. (76) EGFR (Receptor of epidermal growth factor) Nucleotide Access of Genbank No. NM_005228 Genbank version No. NM_005228.3 GI: 41927737 Date of GenBank registration update: September 30, 201201: 47 PM Polypeptide Access of Genbank No. NP_005219 Genbank version No. NP_005219.2 GI: 29725609 Date of GenBank registration update: September 30, 201201: 47 PM Cross-references Dhomen NS., Et al Crit Rev Oncog. 2012; 17 (1): 31- 0 Other information Official Symbol: EGFR Other Pseudonyms: ERBB, ERBB1, HERI, PIG61, MENA Other Designations: homologue of the viral oncogene of avian erythroblastic leukemia (v-erb-b); protein 40 cell growth inhibitor; protein 61 inducer of cell proliferation; proto-oncogene c-ErbB-1; tyrosine kinase protein of erbB-1 receptor ANTIBODIES BMS: Cetuximab (Erbitux) - Broadbridge VT., Et al Expert Rev Anticancer Ther. 2012 May; 12 (5): 555-65.
For example, see US6217866 - ATTC deposit No. 9764.
Amgen: Panitumumab (Vectibix) - Argü is G., et al Future Oncol. 2012 Apr; 8 (4): 373-89 For example, see US6235883 SEQ ID NOs: 23-38. Genmab: Zalutumumab - Rivera F., et al Expert Opin Biol Ther. 2009 May; 9 (5): 667-74.
YM Biosciences: Nimotuzumab - Ramakrishnan MS., Et al MAbs. 2009 Jan-Feb; 1 (1): 41-8.
For example, see US5891996 SEQ ID NOs: 27-34. (77) Her3 (ErbB3) - ERBB3 (homologue 3 of the viral oncogene of the erythroblastic leukemia v-erb-b2 (avian)) Nucleotide Access of Genbank No. M34309 Genbank version No. M34309.1 GI: 183990 Update date of the GenBank registry: June 23, 201008: 47 PM Polypeptide Access of Genbank No. AAA35979 Genbank version No. AAA35979.1 GI: 306841 Update date of GenBank registration: June 23, 201008: 47 PM Cross-references Plowman, G D. , et al Proc. Na ti. Acad. Sci. U. S. A. 87 (13), 4905-4909 (1990) Other information Official Symbol: ERBB3 Other Pseudonyms: ErbB-3, HER3, LCCS2, MDA-BF-1, c-erbB-3, c-erbB3, erbB3-S, pl80-ErbB3, p45-sErbB3, p85-sErbB3 Other Designations: c-ErbB-3 protein similar to proto-oncogene; protein tyrosine kinase erbB-3 receptor; HER3 cell surface tyrosine kinase receptor ANTIBODIES Merimack Pharma: MM-121 (Schoeberl B., et al Cancer Res.2010 Mar 15; 70 (6): 2485-2494) For example, see US2011028129 SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 8. (78) RON-MST1R (Macrophage Stimulant Receptor 1 (tyrosine kinase related to c-met)) Nucleotide Access of Genbank No. X70040 Genbank version No. X70040.1 GI: 36109 Update date of GenBank registration: February 2, 201110: 17 PM Polypeptide Access of Genbank No. CCA49634 Genbank version No. CCA49634.1 GI: 36110 Update date of GenBank registration: February 2, 201110: 17 PM Cross-references Ronsin C., et al Oncogene 8 (5), 1195-1202 (1993) Other information Official Symbol: MST1R Other Pseudonyms: CD136, CDwl36, PTK8, RON Other Designations: MSP recipient; variant of MST1R RON30; variant of MST1R RON62; protein tyrosine kinase 8 PTK8; variant of RON E2E3; tyrosine kinase related to c-met; macrophage stimulating protein receptor; pl85-Ron; variant 1 of soluble RON; variant 2 of soluble RON; 3 variant of soluble RON; variant 4 of soluble RON (79) EPHA2 (Receptor A2 of EPH) Nucleotide Access of Genbank No. BC037166 Genbank version No. BC037166.2 GI: 33879863 Update date of the GenBank registry: 6 March 201201: 59 PM Polypeptide Access of Genbank No. AAH37166 Genbank version No. AAH37166.1 GI: 22713539 GenBank registration update date: March 6, 201201: 59 PM Cross-references Strausberg R.L., et al Proc. Nati Acad. Sel. USES. 99 (26), 16899-16903 (2002) Other information Official Symbol: EPHA2 Other Pseudonyms: ARCC2, CTPA, CTPP1, ECK Other Designations: receiver 2 of ephrin type A; epithelial cell receptor tyrosine kinase protein; variant 1 of soluble EPHA2; ECK receptor tyrosine kinase protein ANTIBODIES Medimmune: 1C1 (Lee JW., Et al Clin Cancer Res. 2010 May 1; 16 (9): 2562-2570) For example, see US20090304721A1 Fig.7 and 8. (80) CD20-MS4A1 (4 domains crossing the membrane, subfamily A, member D Nucleotide Access of Genbank No. M27394 Genbank version No. M27394.1 GI: 179307 Date of GenBank registration update: November 30, 200911: 16 AM Polypeptide Access of Genbank No. AAA35581 Genbank version No. AAA35581.1 GI: 179308 Date of GenBank registration update: November 30, 200911: 16 AM Cross-references Tedder T.F., et al Proc. Nati Acad. Sel. U. S. A. 85 (1), 208-212 (1988) Other information Official Symbol: MS4A1 Other Pseudonyms: Bl, Bp35, CD20, CVID5, LEU-16, MS4A2, S7 Other Designations: lymphocyte CD20 antigen B; Bl cell surface antigen B lymphocytes; CD20 antigen; CD20 receiver; Leu-16 leukocyte surface antigen ANTIBODIES Genentech / Roche: Rituximab - Abdulla NE., Et al BioDrugs. 2012 Apr 1; 26 (2): 71-82.
For example, see US5736137, ATCC deposit No.
HB-69119.
GSK / Genmab: Ofatumumab Nightingale G., et al.
Ann Pharmacother.2011 Oct; 5 (10): 128-55.
For example, see US20090169550A1 SEQ ID NOs: 2, 4 and 5 Immunomedics: Veltuzumab - Goldenberg DM., Et al Leuk Lymphoma. 2010 May; 51 (5): 747-55.
For example, see US7919273B2 SEQ ID NOs: 1, 2, 3, 4, 5 and 6. (8D Tenascin C - TNC (Tenascin C) Nucleotide Access of Genbank No. NM_002160 Genbank version No. NM_002160.3 GI: 340745336 Date of GenBank registration update: September 23, 201202: 33 PM Polypeptide Access of Genbank No. NP_002151 Genbank version No. NP_002151.2 GI: 153946395 Date of GenBank registration update: September 23, 201202: 33 PM Cross-references Nies D.E., et al J. Biol. Chem.266 (5), 2818-2823 (1991); Siri A., et al Nucleic Acids Res.19 (3), 525-531 (1991) Other information Official Symbol: TNC Other Pseudonyms: 150-225, GMEM, GP, HXB, JI TN, TN-C Other Designations: GP 150-225; cytotactin antigen of the extracellular matrix associated with glioma hexabraquione (tenascin); myotendinous antigen neuronectin; tenascin; isoform 14 / AD1 / 16 of tenascin-C ANTIBODIES Philogen: Gil (von Lukowicz T., et al J Nucí Mecí, 2007 Apr; 48 (4): 582-7) and F16 (Pedretti M. et al Lung Cancer, 2009 Apr; 64 (1): 28-33) For example, see US7968685 SEQ ID NOs: 29, 35, 45 and 47. (82) FAP (fibroblast activation protein, alpha) Nucleotide Access of Genbank No. U09278 Genbank version No. U09278.1 GI: 1888315 Update date of GenBank registration: June 23, 201009: 22 AM Polypeptide Access of Genbank No. AAB49652 Genbank version No. AAB49652.1 GI: 1888316 Date of GenBank registration update: June 23, 201009: 22 AM Cross-references Scanlan, M.J., et al Proc. Nati Acad. Sci.
U. S. A. 91 (12), 5657-5661 (1994) Other information Official Symbol: FAP Other Pseudonyms: DPPIV, FAPA Other Designations: gelatinase bound to melanoma membrane of 170 kDa; integral membrane serine protease; seprasa (83) DKK-1 (Homologue of Dickkopf 1 (Xenopus laevis)) Nucleotide Access of Genbank No. NM_012242 Genbank version No. NM_012242.2 GI: 61676924 Date of GenBank registration update: September 30, 201201: 48 PM Polypeptide Access of Genbank No. NP_036374 Genbank version No. NP_036374.1 GI: 7110719 Date of GenBank registration update: September 30, 201201: 48 PM Cross-references Fedi P. et al J. Biol. Chem. 274 (27), 19465-19472 (1999) Other information Official Symbol: DKK1 Other Pseudonyms: UNQ492 / PR01008, DKK-1, SK Other Designations: protein 1 related to dickkopf; similar to dickkopf 1; protein 1 similar to dickkopf; protein 1 related to dickkopf; hDkk-1 ANTIBODIES Novartis: BHQ880 (Fulciniti M., et al Blood, 2009 Jul 9; 114 (2): 371-379) For example, see US20120052070A1 SEC ID NOs: 100 and 108. (84) CD52 (Molecule CD52) Nucleotide Access of Genbank No. NM_001803 Genbank version No. NM_001803.2 GI: 68342029 Date of GenBank registration update: September 30, 201201: 48 PM Polypeptide Access of Genbank No. NP_001794 Genbank version No. NP_001794.2 GI: 68342030 Date of GenBank registration update: September 0, 201201: 48 PM Cross-references Xia M.Q., et al. Eur. J. Immunol. 21 (7), 1677- 684 (1991) Other information Official Symbol: CD52 Other Pseudonyms: CDW52 Other Designations: antigen CAMPATH-1; antigen CD52 (CAMPATH-1 antigen); antigen CDW52 (antigen CAMPATH-1); antigen 1 of the pathology of Cambridge; epididymal secretory E5 protein; he5; protein 5 specific for human epididymis ANTIBODIES Alemtuzumab (Ca path) - Skoetz N., et al Cochrane Database Syst Rev. 2012 Feb 15; 2: CD008078.
For example, see access to DrugBank No. DB00087 (BIOD00109, BTD00109) (85) CS1 - SLAMF7 (Member 7 of the family of SLAM) Nucleotide Access of Genbank No. NM_021181 Genbank version No. NM_021181.3 GI: 1993571 Date of GenBank registration update: June 29, 201211: 24 AM Polypeptide Access of Genbank No. NP__06700 Genbank version No. NP_067004.3 GI: 19923572 GenBank registration update date: June 29, 201211: 24 AM Cross-references Boles K.S., et al Immunogenetics 52 (3-4), 302- 07 (2001) Other information Official Symbol: SLAMF7 Other Pseudonyms: UNQ576 / PR01138, 19A, CD319, CRACC, CS1 Other Designations: protein 19A24; subset 1 of CD2; cytotoxic cells activating the CD2-like receptor; cytotoxic cells activating the CD2-like receptor; FOAP-12 membrane protein; protein similar to novel LY9 (antigen 9 of lymphocytes); 19A protein ANTIBODIES BMS: elotuzumab / HuLuc63 (Benson DM., Et al J Clin Oncol, 2012 Jun 1; 30 (16): 2013-2015) For example, see US20110206701 SEC ID NOs: 9, 10, 11, 12, 13, 14, 15 and 16. (86) Endoglina - ENG (Endoglina) Nucleotide Access of Genbank No. AF035753 Genbank version No. AF035753.1 GI: 3452260 Date of GenBank registration update: March 10, 201006: 36 PM Polypeptide Access of Genbank No. AAC32802 Genbank version No. AAC32802.1 GI: 3452261 Update date of the GenBank registry: March 10, 201006: 36 PM Cross-references Rius C., et al Blood 92 (12), 4677-4690 (1998) Official Symbol: ENG Other information Other Pseudonyms: RP11-228B15.2, CD105, END, HHT1, ORW, ORW1 Other Designations: CD105 antigen (87) Annexin Al - ANXA1 (Annexin Al) Nucleotide Access of Genbank No. X05908 Genbank version No. X05908.1 GI: 34387 GenBank registration update date: February 2, 201110: 02 AM Polypeptide Access of Genbank No. CCA29338 Genbank version No. CCA29338.1 GI: 34388 Update date of GenBank registration: February 2, 201110: 02 AM Cross-references Wallner B.P., et al Nature 320 (6057), 77-81 (1986) Other information Official Symbol: ANXA1 Other Pseudonyms: RP11-71A2 .1, ANX1, LPC1 Other Designations: annexin I (lipocortin I); annexin-1; Calpactin II; calpactin-2; chromobindin-9; lipocortin I; p35; phospholipase A2 inhibitor protein (88) V-CAM (CD106) - VCAM1 (Molecule 1 of adhesion to vascular cells) Nucleotide Access of Genbank No. M60335 Genbank version No. M60335.1 GI: 340193 Date of GenBank registration update: June 23, 201008: 56 AM Polypeptide Access of Genbank No. AAA61269 Genbank version No. AAA61269.1 GI: 340194 Update date of GenBank registration: June 23, 201008: 56 AM Cross-references Hession C., et al J. Biol. Chem. 266 (11), 6682-6685 (1991) Other information Official Symbol VCAM1 Other Pseudonyms: CD106, INCAM-100 Other Designations: CD106 antigen; protein 1 of adhesion to vascular cells Antibody Sequences Anti-Integrina anb6 RHAB6.2 QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTE YAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTAVPNLRGDLQVLAQKVAG PYPFDYWGQGTLVTVSS RHCB6. 2 QVQLVQSGAEVKKPGASVKVSCKASGYTFIDSYMHWVRQAPGQRLEWMGWIDPENGDTE YAPKFQGRVTITTDTSASTAYMELSSLRSEDTAVYYCARGTPTAVPNLRGDLQVLAQKVAG PYPFDYWGQGTLVTVSS RHF QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDT EYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTGPYYFDYWGQGTLVTV H.H RHFB6 QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDT EYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTAVPNLRGDLQVLAQKVA GPYYFDYWGQGTLVTVSS RHAYlOObP QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTE YAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTGPYPFDYWGQGTLVTVSS RKF ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKFL36L50 ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWLQQKPGQAPRLLIYLTSNLASGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKC EIVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFS GSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK An ti -CD33 CD33 Huml95 VH QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTG YNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS CD33 Huml95 VK DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSG VPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK Anti-CD19 VH surface conditioned from CD19 B4 QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYTN YNQNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSVTV SS VK surface conditioned from CD19 B4 EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPAR FSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK Anti -Her2 Herceptin VH chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY1HWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS S VL chain of Herceptin DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK Anti-CD25 VK of Simulect (also known as Basiliximab) QIVSTQSPAIMSASPGEKV CSASSSRSYMQWYQQKPGTSPKRWIYDTSKLASGVPAR FSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYTFGGGTKLEIK VH of Simulect QLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQRPGQGLEWIGAIYPGNSDTSYN QKFEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSRDYGYYFDFWGQGTTLTVSS Anti-PSMA VH '1 deimmunized EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYT1HWVKQAPGKGLEWIGNINPNNGGTTYN QKFEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSS VK '1 deimmunized DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYWASTRHTGIPSR FSGSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIK VH1 '5 deimmunized EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMN VRQAPGKGLEWVAEIRSQSNNF ATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTGVYYCTRRWNNFWGQGTTVTVSS VH2 '5 deimmunized EVKLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFA THYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS VH3 '5 deimmunized EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFA THYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS VH4 '5 deimmunized EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFA THYAESVKGRFTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS VK1 '5 deimmunized NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPD RFTGSGSATDFTLTISSLQTEDLADYYCGQSYTFPYTFGQGTKLEMK VK2 '5 deimmunized NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPD RFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK VK3 '5 deimmunized NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPD RFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK VK4 '5 deimmunized NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPD RFSGSGSGTDFTLTISSLQAEDEADYYCGQSYTFPYTFGQGTKLEIK VK DI '5 deimmunized NIVMTQFPKSMSASAGERMTLTCKASENVGTYVS YQQKPTQSPKMLIYGASNRFTGVPD RFSGSGSGTDFILTISSVQAEDLVDYYCGQSYTFPYTFGGGTKLEMK VH DI '5 deimmunized EVKLEESGGGLVQPGGSMKISCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRSQSNNFA THYAESVKGRVIISRPPSKSSVYLQMNSLRAEPTAVYYCTRRWNNFWGQGTTVTVSS RHA '5 humanized EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFA THYAESVKGRFTISRPPSKNTAYLQMNSLKTEPTAVYYCTRRWNNFWGQGTTVTVSS RHB '5 humanized EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRVIISRPPSKNTVYLQMNSLRTEPTAVYYCTRRWNNFWGQGTTVTVSS RHC '5 humanized EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRVIISRPPSKNTVYLQMNSLRTEPTAVYYCTRRWNNFWGQGTTVTVSS RHD '5 humanized EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFA THYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS RHE 15 humanized EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRFTISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS RHF '5 humanized EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFGQGTTVTVSS RHG '5 humanized EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRR NNFWGQGTTVTVSS RKA '5 humanized DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSR FSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK RKB '5 humanized DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSR FSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK RKC '5 humanized DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLI YGASNRFTGVPS RFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK RKD '5 humanized DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPS RFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK RKE '5 humanized NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPDR FTGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK RKF '5 humanized NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSR FSGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK RKG '5 humanized NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPDR FTGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK The parental antibody can also be a fusion protein comprising a sequence of albumin binding peptides (ABP) (Dennis et al (2002) "Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins" J Biol Chern. 277: 35035-35043; WO 01/45746). The antibodies of the invention include fusion proteins with ABP sequences taught by: (i) Dennis et al (2002) J Biol Chem. 277: 35035-35043 in Tables III and IV, page 35038; (ii) US 2004/0001827 in
[0076]; and (iii) WO 01/45746 on pages 12-13, which are all incorporated herein by reference.
In one embodiment, the antibody has been produced to specifically target the antigen related to the tumor or? nbb · The cell-binding agent can be labeled, for example, to aid in the detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate. The brand can be a biotin brand. In another embodiment, the cell binding agent can be labeled with a radioisotope.
The embodiments of the present invention include ConjA wherein the cell-binding agent is selected from an antibody for any of the antigens discussed above.
Modes of the present invention include the ConjB wherein the cell-binding agent is selected from an antibody for any of the antigens discussed above.
The embodiments of the present invention include ConjA wherein the cell-binding agent is selected from any of the antibodies discussed above.
The embodiments of the present invention include the ConjB wherein the cell-binding agent is selected from any of the antibodies discussed above.
The present invention also relates to conjugates wherein the cell-binding agent is selected from an antibody for any of the antigens discussed above and any of the antibodies discussed above linked to different drugs.
Drug loading The drug load is the average number of PBD drugs per cell-binding agent, e.g., antibody. If the compounds of the invention are bound to cysteines, the drug loading can vary from 1 to 8 drugs (D) per cell-binding agent, i.e., wherein 1, 2, 3, 4, 5, 6, 7 and 8 drug portions are covalently linked to the cell binding agent. Conjugate compositions include collections of cell binding agents, eg, antibodies, conjugated with a range of drugs, from 1 to 8. If the compounds of the invention are bound to lysines, the drug loading may vary from 1 to 80 drugs (D) per cell-binding agent, although an upper limit of 40, 20, 10 or 8 may be preferred. Conjugate compositions include collections of cell binding agents, eg, antibodies, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.
The average number of drugs per antibody in ADC preparations of conjugation reactions can be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, assay of ELISA, and electrophoresis. The quantitative distribution of ADC can also be determined in terms of p. By ELISA, the averaged p-value in a particular ADC preparation can be determined (Hamblett et al (2004) Clin Cancer Res. 10: 7063-7070; Sanderson et al (2005) Cancer Clin. Res. 11: 843- 852). However, the distribution of p (drug) values is not discernible by antibody-antigen binding and the limitation of ELISA detection. In addition, the ELISA assay for the detection of antibody-drug conjugates does not determine whether the drug portions are bound to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues. In some cases, separation, purification and characterization of homogeneous ADCs can be achieved where p is a certain ADC value with other drug loads by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.
For some antibody-drug conjugates, p may be limited by the number of binding sites in the antibody. For example, an antibody can have only one or several thiol groups of cysteine, or it can have only one or several sufficiently reactive thiol groups through which a linker can be attached. A higher drug load, for example, p > 5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates.
Normally, less than the theoretical maximum of the drug portions are conjugated with an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate (D-L) or linker reagent. Only the more reactive lysine groups can react with a linker reagent that is reactive with amine. In addition, only the more reactive thiol cysteine groups can react with a linker reagent that is thiol reactive. Generally, antibodies do not contain many, if any, free thiol groups of cysteine and reagents that can bind to a drug moiety. Most thiol residues of cysteine in the antibodies of the compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions. The loading (drug / antibody ratio) of an ADC can be controlled in a number of different ways, including: (i) limiting the molar excess of the drug-linker intermediate (DL) or linker reagent with respect to the antibody, (ii) limit the reaction time of the conjugation or temperature, and (iii) partial reducing or limiting reducing conditions for the modification of the cysteine thiol.
Certain antibodies have disulfides between reducible chains, that is, cysteine bridges. The antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the U-syna reaction with 2-iminothiolane (Traut's reagent) which results in the conversion of an amine to a thiol. The reactive thiol groups can be introduced into the antibody (or fragment thereof) by manipulating one, two, three, four or more cysteine residues (for example, by preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). US 7521541 teaches to manipulate antibodies by the introduction of reactive cysteine amino acids.
Cysteine amino acids can be manipulated at sites reactive in an antibody and do not form interchain or intermolecular disulfide bonds (Junutula et al., 2008b Nature Biotech., 26 (8): 925-932; Dom an et al (2009) Blood 114 (13): 2721-2729; US documents 7521541; US 7723485; W02009 / 052249). The manipulated cysteine thiols can react with linker reagents or the drug-linker reagents of the present invention having thiol-reactive electrophilic groups such as maleic ida or alpha-haloamides to form ADC with antibodies manipulated by cysteine and the drug portions of PBD. In this way, the location of the drug portion can be designed, controlled and known. The drug loading can be controlled, since the manipulated cysteine thiol groups usually react with thiol-reactive linker reagents or drug-linker reagents in high yield. The manipulation of an IgG antibody to introduce a cysteine amino acid by substitution at a single site in the heavy or light chain gives two new cysteines in the symmetric antibody. A drug loading close to 2 can be achieved with near homogeneity of the ADC conjugation product.
If more than one nucleophilic or electrophilic group of the antibody reacts with a drug-linker intermediate, or linker reagent followed by drug portion reagent, then the resulting product is a mixture of ADC compounds with a distribution of drug portions attached to the drug. an antibody, for example, 1, 2, 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) can separate compounds in the mixture by the load value of the drug. ADC preparations with a single drug loading value (p) can be isolated, however, these single charge value ADCs can still be heterogeneous mixtures because the drug moieties can be linked, via the linker, in different places in the antibody.
In this manner, the antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds wherein the antibody has one or more drug portions of PBD and wherein the drug portions can be bound to the antibody in various amino acid residues.
In one embodiment, the average number of dimeric pyrrolobenzodiazepine groups per cell-binding agent is in the range 1 to 20. In some embodiments, the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4. , and 4 to 8.
In some embodiments, there is a dimeric pyrrolobenzodiazepine group per cell binding agent.
Includes Other Forms Unless otherwise specified, the above includes the ionic, salt, solvate and well-known protectors of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic form (carboxylate) (-COCT), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N + HR1R2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0), a salt or solvate thereof, as well as conventional protected forms.
You go out It may be convenient or desirable to prepare, purify and / or handle a corresponding salt of the active compound, for example, a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al. , J. Pharm. Sci. , 66, 1-19 (1977).
For example, if the compound is anionic, or has a functional group that can be anionic (for example, -COOH can be -COO), then a salt with a suitable cation can be formed. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K +, alkaline earth cations such as Ca2 + and Mg2 +, and other cations such as Al + 3. Examples of suitable organic cations include, but are not limited to, ammonium ion (ie, NH4 +) and substituted ammonium ions (eg, NH3R +, NH2R2 +, NHR3 +, NR4 +). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as U sine and Arginine An example of a common quaternary ammonium ion is N (CH3) 4+.
If the compound is cationic, or has a functional group that can be cationic (for example, -NH2 can be -NH3 +), then a salt with a suitable anion can be formed. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitroso, phosphoric, and phosphorous.
Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, emetic, ethanedisulfonic, ethanesulfonic, fumaric, gluteonic, gluconic, glutamic, glycolic, hydroxylamino, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, mellic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulphanilic, tartaric, toluenesulfonic, trifluoroacetic and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose.
Solvates It may be convenient or desirable to prepare, purify and / or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a solute complex (e.g., active compound, salt of active compound) and solvent. If the solvent is water, the solvate can conveniently be referred to as a hydrate, for example, a monohydrate, a dihydrate, a trihydrate, etc.
The invention includes compounds wherein a solvent is added through the imine bond of the PBD portion, which is illustrated below, wherein the solvent is water or an alcohol (RAOH, where RA is Ci_4 alkyl): These forms can be referred to as carbinolamine and carbinolamine ether forms of the PBD (as described in the section relating to R10 above). The balance of these equilibria depends on the conditions to which the compounds are found, as well as the nature of the portion itself.
These particular compounds can be isolated in solid form, for example, by lyophilization.
Isomers Certain compounds of the invention may exist in one or more particular geometrical, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational or anomeric forms, including, but not limited to, cis and trans forms; E and Z forms; forms c, t and r; endo and exo forms; R, S and meso forms; D and L forms, forms d and 1; forms (+) and (-); keto, enol and enolate forms; Sin and anti forms; synclinal and antialinal forms; forms a and b; axial and equatorial forms; forms of boat, chair, turn, envelope and half chair; and combinations thereof, hereinafter collectively referred to as "isomers" (or "forms") isomeric ").
The term "chiral" refers to molecules that have the property of non-overlapping the component of the mirror image, while the term "achiral" refers to molecules that are superimposable on their mirror image component.
The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ with respect to the arrangement of atoms or groups in space.
"Diastereomer" refers to a stereoisomer with two or more centers that chirality and whose molecules are not mirror images of each other. The diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography.
"Enantiomers" refer to two stereoisomers of a compound that are mirror images not superimposable with each other.
The stereochemical definitions and conventions used in the present document generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wilcy & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including, but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of the polarized plane-light. In the description of an optically active compound, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the molecule around its chiral center (s). The prefixes d and I or (+) and (-) are used to designate the sign of rotation of the plane-light polarized by the compound, meaning (-) or I that the compound is levorotatory. A compound with the prefix (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical, except that they are mirror images of each other. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers refers to a racemic mixture or a racemate, which may occur if there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, which lack the optical activity.
Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (ie, isomers that differ in the connections between atoms instead of simply by the position of atoms 40 in space). For example, a reference to a methoxy group, -OCH3, should not be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may include structurally isomeric forms that fall within that class (eg, Ci-7 alkyl includes n-propyl and iso-propyl, butyl includes n-, iso-, sec and tere-butyl; methoxyphenyl includes ortho-, meta- and para-methoxyphenyl).
The above exclusion does not refer to forms tautomers, for example, keto, enol and enolate forms, as in, for example, the following tautomeric pairs: keto / enol (illustrated below), imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enetiol, N-nitroso / hydroxyazo and nitro / aci-nitro. enol enolate The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible by means of a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions through the migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by rearrangement of some of the binding electrons.
Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, which includes 1H, 2H (D), and 3H (T); C can be in any isotopic form, which includes 12C, 13C, and 14C; Or it can be in any isotopic form, which includes 160 and 180; and similar.
Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to 2H (deuterium, D), 3H (tritium) , nC, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36C1, and 125I. Various isotopically-labeled compounds of the present invention, for example, those in which radioactive isotopes such as 3H, 13C, and 14C are incorporated. Such isotopically-labeled compounds may be useful in metabolic studies, reaction kinetics studies, detection techniques or imaging, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including distribution assays. drug tissue or substrate, or in the radioactive treatment of patients. The therapeutic compounds labeled or substituted with deuterium of the invention may have improved DMPK properties (drug metabolism and pharmacokinetics), relating to distribution, metabolism, and secretion (ADME). Substitution with heavier isotopes such as deuterium may provide certain therapeutic advantages resulting from increased metabolic stability, for example, elevated in vivo half-life or reduced dosage requirements. A compound labeled with 18F may be useful for PET or SPECT studies. The compounds isotopically Labels of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and the preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. Additionally, replacement with heavier isotopes, particularly deuterium (ie, 2H or D), may provide certain therapeutic advantages resulting from increased metabolic stability, for example, elevated in vivo half-life or reduced dosage requirements or an improvement in the index. therapeutic. It is understood that deuterium in this context is considered a substituent. The concentration of such heavier isotope, specifically deuterium, can be defined by an isotopic enrichment factor. In the compounds of the present invention, any atom not specifically designated as a particular isotope is understood to represent any stable isotope of that atom.
Unless otherwise specified, a reference to a particular compound includes all those isomeric forms, which include (fully or partially) racemic mixtures and other mixtures thereof. Methods for preparation (eg, asymmetric synthesis) and separation (eg, crystallization fractionated and chromatographic means) of such isomeric forms are either known in the art, or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
Biological Activity In vitro cell proliferation assays Generally, the cytotoxic or cytostatic activity of an antibody-drug conjugate (ADC) is measured by: exposure of mammalian cells having receptor proteins, eg, HER2, to the ADC antibody in a cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and the measurement of cell viability. Cell-based in vi tro assays are used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (activation by caspases) of an ADC of the invention.
The in vitro potency of antibody-drug conjugates can be measured by a cell proliferation assay. The CellTiter-Glo® Luminescent Cell Viability Assay is a commercially available homogeneous assay method (Promega Corp., Madison, WI) based on the recombinant expression of luciferase from coleoptera (US Patent Nos.5583024; 5674713 and 5700670). This cell proliferation assay determines the number of viable cells in culture based on the quantification of ATP present, an indicator of metabolically active cells (Crouch et al (1993) J. Immunol.Meth.160: 81-88; US 6602677; ). The CellTiter-Glo® Assay is performed in a 96 well format, making it susceptible to automated high resolution screening (HTS) (Cree et al (1995) AntiCancer Drugs 6: 398-404). The homogeneous assay procedure involves the addition of the single reagent (CellTiter-Glo® Reagent) directly to cells grown in medium supplemented with serum. Cell washing, elimination of medium and multiple stages of pipetting are not required. The system detects only 15 cells / well in a 384-well format in 10 minutes after the addition of reagent and mixing. The cells can be treated continuously with ADC, or they can be treated and separated from ADC. Generally, cells treated briefly, ie, 3 hours, showed the same potency effects as continuously treated cells.
The homogeneous "add-mix-measure" format results in cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The CellTiter-Glo® Assay generates a "glow-type" luminescent signal, produced by the luciferase reaction, which has a half-life generally greater than five hours, depending on the type of cell and the medium used. Viable cells are reflected in relative units of luminescence (URL). The substrate, Beetle Luciferin, is oxidatively decarboxylated by recombinant firefly luciferase, with the concomitant conversion of ATP to A P and generation of photons.
The in vitro potency of antibody-drug conjugates can also be measured by a cytotoxicity assay. Adherent cells grown with PBS are washed, detached with trypsin, diluted in complete medium containing 10% FBS, centrifuged, resuspended in fresh medium and counted with a hemocytometer. Suspended crops are counted directly. The onodisperse cell suspensions suitable for counting may require agitation of the suspension by repeated aspiration to break the cell groups.
The cell suspension is diluted to the desired seed density and dispensed (100 ml per well) into 96-well black plates. Adherent cell line plates are incubated overnight to Allow adhesion. Cell cultures in suspension can be used on the day of sowing.
A stock solution (1 ml) of ADC (20 pg / ml) is prepared in the appropriate cell culture medium. Serial 10-fold dilutions of stock solution ADC are prepared in 15 ml centrifuge tubes by serially transferring 100 ml to 900 ml of cell culture medium.
Four wells are dispensed in duplicate of each dilution of ADC (100 μm) in 96-well black plates, previously seeded with cell suspension (100 μm), resulting in a final volume of 200 μm. The control wells receive cell culture medium (100 μm).
If the doubling time of the cell line is greater than 30 hours, the incubation of ADC is for 5 days, if a four-day incubation is not done.
At the end of the incubation period, cell viability is evaluated with the Alamar blue test. Blue Alamar (Invitrogen) is dispensed over the entire plate (20 μm per well) and incubated for 4 hours. The fluorescence of Alamar blue is measured at the excitation of 570 nm, emission of 585 nm in the Varioskan Flash plate reader. The percentage of cell survival is calculated from the mean fluorescence in the wells treated with ADC compared to the average fluorescence in the wells of control.
Efficacy in vivo The in vivo efficacy of the antibody-drug conjugates (ADCs) of the invention can be measured by tumor xenograft studies in mice. For example, the in vivo efficacy of an anti-HER2 ADC of the invention can be measured by a high expression transgene explant HER2 mouse model. An allograft of the transgenic mouse Fo5 ramtv is propagated that does not respond to, or responds poorly to, therapy with HERCEPTIN®. Subjects are treated once with ADC at certain dose levels (mg / kg) and exposure to the PBD drug (pg / m2); and control with placebo buffer (Vehicle) and monitored for two weeks or more to measure the time to tumor duplication, the logarithm of cell destruction and tumor shrinkage.
Use The conjugates of the invention can be used to provide a PBD compound at a target location.
The target location is preferably a population of proliferating cells. The antibody is an antibody to an antigen present in a population of proliferating cells.
In one embodiment, the antigen is absent or present at a reduced level in a population of non-proliferating cells compared to the amount of antigen present in the population of proliferating cells, for example, a population of tumor cells.
At the target location, the linker can be cleaved in order to release a RelA or RelB compound. In this manner, the conjugate can be used to selectively provide a RelA or RelB compound to the target location.
The linker can be cleaved by an enzyme present at the target location.
The target location can be in vitro, in vivo or ex vivo.
The antibody-drug conjugate (ADC) compounds of the invention include those with utility for anti-cancer activity. In particular, the compounds include a conjugated antibody, i.e., covalently linked via a linker, to a drug portion of PBD, i.e., toxin. If the drug is not conjugated to an antibody, the PBD drug has a cytotoxic effect. The biological activity of the drug portion of PBD is thus modulated by conjugation with an antibody. The antibody-drug conjugates (ADCs) of the invention selectively deliver an effective dose of a cytotoxic agent to tumor tissue, so that it can be achieve greater selectivity, that is, a lower effective dose.
Thus, in one aspect, the present invention provides a conjugate compound as described herein for use in therapy.
In a further aspect, a conjugate compound as described herein is also provided for use in the treatment of a proliferative disease. A second aspect of the present invention provides the use of a conjugate compound in the manufacture of a medicament for the treatment of a proliferative disease.
One of ordinary skill in the art can easily determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays that can be conveniently used to evaluate the activity offered by a particular compound are described in the following examples.
The term "proliferative disease" refers to undesired or uncontrolled cell proliferation of excessive or abnormal cells that is undesirable, such as neoplastic or hyperplastic growth, either in vi tro or in vi vo.
Examples of proliferative conditions include, but are not limited to, benign, pre malignant and malignant cell proliferation, including but not limited to, neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g., lung cancer, lung cancer). small cells, gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias, psoriasis, bone diseases, fibroproliferative disorders (for example, connective tissues), and atherosclerosis. Cancers of particular interest include, but are not limited to, leukemias and ovarian cancers.
Any type of cell can be treated, including, but not limited to, lung, gut or intestinal (including, for example, bowel, colon), breast (mammary), ovary, prostate, liver (hepatic), kidney (kidney) ), bladder, pancreas, brain, and skin.
In one modality, the treatment is a pancreatic cancer.
In one embodiment, the treatment is from a tumor that has integrin anb6 on the surface of the cell.
It is contemplated that antibody-conjugates Drug (ADC) of the present invention can be used to treat various diseases or disorders, for example, characterized by overexpression of a tumor antigen. Exemplary hyperproliferative conditions or disorders include benign or malignant tumors; leukemia, malignant haematological and lymphoid tumors. Others include neuronal, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stromal, blastocoelic, inflammatory, angiogenic, and immunological disorders, including autoimmune.
Generally, the disease or disorder that will be treated is a hyperproliferative disease such as cancer. Examples of the cancer that will be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoid tumors. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer that includes small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung. , cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, cancer liver, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, carcinoma of the salivary glands, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer , hepatic carcinoma, anal carcinoma, penile carcinoma, as well as cancer of the head and neck.
Autoimmune diseases for which the ADC compounds can be used in the treatment include rheumatological disorders (such as, for example, rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis / dermatomyositis, cryoglobulinemia, antiphospholipid antibodies and psoriatic arthritis), osteoarthritis, gastrointestinal and liver autoimmune disorders (such as, for example, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis , primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteritis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opsoclonus-myoclonus syndrome, myasthenia gravis, neuromyelitis optics, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), kidney disorders (such as, for example, glomerulonephritis, Goodpasture syndrome and Berger's disease), autoimmune dermatological disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, huffy pemphigoid, and cutaneous lupus erythematosus), hematological disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune auditory diseases (such as such as, for example, inner ear disease and partial deafness), Behcet's disease, Raynaud's syndrome, organ transplantation, and autoimmune endocrine disorders (such as, for example, autoimmune diseases related to diabetes such as insulin-dependent diabetes mellitus. (DMDI), Addison's disease, and autoimmune thyroid disease ne (for example, Graves disease and thyroiditis)). Such more preferred diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.
Treatment Methods The conjugates of the present invention can be used in a therapy method. A method of treatment is also provided, which comprises administering to a subject in need of treatment a therapeutically effective amount of a conjugate compound of the invention. The term "therapeutically effective amount" is an amount sufficient to show benefit to a patient. Such benefit may be at least the improvement of at least one symptom. The actual amount administered, and the speed and time-cycle of administration, will depend on the nature and severity of what is being treated. The prescription of treatment, for example, decisions about the dosage, is within the responsibility of general practitioners and other medical doctors.
A compound of the invention can be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, for example, drugs, such as chemotherapeutics); surgery; and radiotherapy.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of the mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spider venom alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy.
Examples of chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech / OSI Pharm.), Docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine ( GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum (II), CAS No.15663-27-1), carboplatin (CAS No. 41575- 94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo) [4.3.0] nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plow), tamoxifen ((Z) -2- [4- ( 1,2-diphenylbut-1-enyl) phenoxy] -NjIV-dimethylethanamine, N0LVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD, and rapamycin.
More examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), Sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma , Astra Zeneca), SF-1126 (inhibitor of PI3K, Semafore Pharmaceuticals), BEZ-235 (inhibitor of PI3K, Novartis), XL-147 (inhibitor of PI3K, Exelixis), PTK787 / ZK 222584 (Novartis), fulvestrant (FASLODEX ®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR ™, SCH 66336, Schering Plow), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA ™, Johnson &Johnson), ABRAXANE ™ (without Cremophor), manipulated nanoparticle formulations with paclitaxel albumin (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG157 1 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmíthKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); alkylsulfonates such as busulfan, improsulphan and piposulfane; aziridines such as benzodopa, carbocuone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including the synthetic analog topotecan); Bryostatin; Callistatin; CC-1065 (including its synthetic analogs of adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictine; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediin antibiotics (eg, calicheamicin, gamma calicheamicin II, omega 11 calicheamicin (Angew Chem. Intl. Ed. Engl. (1994) 33: 183-186), dinemycin, dynemycin A, bisphosphonates, such as clodronate A esperamycin, as well as neocarzinostatin chromophore and chromoprotein-related antibiotic ependylin chromophores), aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxidoxorubicin), epirubicin, esububicin, idarubicin, nemorubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin , olivomycins, peplomycin, porphyromycin, puromycin, chelamicin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, tiamiprin, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocythabin, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergic agents such as aminoglutethimide, mitotane, trilostane; folic acid enhancer such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabuchil; bisantrene; edatraxate; defofamin; demecolcine; diazicuone; elfornitin; eliptinium acetate; an epothilone; etoglucid; gallium nitrate, hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofirano; spirogermanium; tenuazonic acid; triazicuone; 2,2 ', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; Dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacitosina; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; 6-thioguanine; ercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; Daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.
Also included in the definition of "chemotherapeutic agent" are: (i) antihormonal agents that act to regulate or inhibit the action of hormones on tumors such as antiestrogens and receptor modulators; selective estrogens (SERMs), which include, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, cheoxifen, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the aromatase enzyme, which regulates the production of estrogens in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® ( exemestane; Pfizer), formestadium, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole, Novartis), and ARIMIDEX® (anastrozole, AstraZeneca); (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (an analogue of 1,3-dioxolane nucleoside cytosine); (iv) inhibitors of protein kinases such as MEK inhibitors (WO 2007/044515); (v) lipid kinases inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, eg, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as inhibitors of VEGF expression (eg, ANGIOZYME®) and inhibitors of HER2 expression; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.
Therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech), are also included in the definition of "chemotherapeutic agent"; cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idee), ofatumumab (ARZERRA®, GSK), pertuzumab (PERJETA ™, OMNITARG ™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab ( Bexxar, Corixia), and the antibody-drug conjugate gemtuzumab ozogamicin (MYLOTARG®, Wyeth).
Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the invention include: ale tuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab, ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab , tacatuzumab tetraxetane, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab tucotuzumab celmoleucine, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.
The pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e., a conjugate compound, an excipient, carrier, buffer, stabilizer or other pharmaceutically acceptable materials either known to those skilled in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The precise nature of the vehicle or other material will depend on the route of administration, which may be oral, or by injection, for example, cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in the form of a tablet, capsule, powder or liquid. A tablet can understand a solid vehicle or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other solution of saccharides or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such as a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the affliction site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is free of pyrogens and has adequate pH, isotonicity and stability. Those of ordinary skill in the art are perfectly capable of preparing suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactate Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required.
Formulations Although it is possible to use the conjugate compound (eg, administered) alone, it is often preferable that it be present as a composition or formulation.
In one embodiment, the composition is a pharmaceutical composition (eg, formulation, preparation, medicament) comprising a conjugate compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
In one embodiment, the composition is a pharmaceutical composition comprising at least one conjugate compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but are not limited to vehicles, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (eg wetting agents), masking agents, coloring agents, flavoring agents and pharmaceutically acceptable sweetening agents. .
In one embodiment, the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.
The vehicles, diluents, suitable excipients, etc., can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Reminqton's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.
Another aspect of the present invention relates to methods of preparing a pharmaceutical composition comprising mixing at least one radiolabelled conjugate with [nC] or conjugate-like compound, as defined herein, together with one or more of other pharmaceutically acceptable ingredients well known to those skilled in the art, for example, carriers, diluents, excipients, etc. If formulated as discrete units (eg, tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.
The term "pharmaceutically acceptable", as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact. with the tissues of the subject in question (eg, human) without excessive toxicity, irritation, allergic response, or other problem or complication, proportional to a reasonable benefit / risk ratio. Each vehicle, diluent, excipient, etc., must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.
The formulations can be prepared by any method well known in the pharmacy technique. Such methods include the step of bringing the active compound into association with a vehicle that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with vehicles (e.g., liquid carriers, finely divided solid carrier, etc.), and subsequently molding the product, if necessary.
The formulation can be prepared to provide fast or slow release; immediate, delayed, controlled or sustained release; or a combination thereof.
Formulations suitable for parenteral administration (eg, by injection), include sterile, pyrogen-free, isotonic, aqueous or non-aqueous liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise otherwise (for example, in a liposome or other microparticle). Such liquids may additionally contain other ingredients pharmaceutically acceptable, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant body fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic vehicles suitable for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactate Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng / ml to about 10 mg / ml, for example, from about 10 ng / ml to about 1 pg / ml. The formulations can be presented in sealed unit dose or multi-dose containers, for example, ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition that requires only the addition of the sterile liquid carrier, eg, water. for injections, immediately before use. Solutions and suspensions for extemporaneous injection can be prepared from sterile powders, granules and tablets.
Dosage It will be appreciated by an expert in the art that the Appropriate dosages of the conjugate compound, and compositions comprising the conjugate compound, may vary from patient to patient. Determining the optimal dosage will usually involve balancing the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on several factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of elimination of the compound, the duration of the treatment, other drugs, compounds , and / or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and previous medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinical professional, although generally the dosage will be selected to achieve local concentrations at the site of action that achieve the desired effect without causing harmful or harmful side effects. substantial Administration can be effected in one dose, continuously or intermittently (eg, in divided doses at appropriate intervals) during the course of treatment. The methods of determining the most effective means and dosage of the administration are well known to those skilled in the art and will vary with the formulation used for the therapy, the purpose of the therapy, the target cell (s) to be treated, and the subject it will be treated. Individual or multiple administrations can be carried out with the level and dose pattern that will be selected by the practical physician, veterinarian or clinical professional.
In general, a suitable dose of active compound is in the range of about 100 ng to about 25 mg (more usually from about 1 pg to about 10 mg) per kilogram of subject body weight per day. If the active compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated based on the parent compound and thus the actual weight that will be used is proportionally increased.
In one embodiment, the active compound is administered to a human patient in accordance with the following dosage regimen: approximately 100 mg, 3 times a day.
In one embodiment, the active compound is administered to a human patient in accordance with the following dosage regimen: approximately 150 mg, twice a day.
In one embodiment, the active compound is administered to a human patient in accordance with the following dosing regimen: approximately 200 mg, twice a day.
However in one embodiment, the conjugate compound is administered to a human patient in accordance with the following dosage regimen: about 50 or about 75 mg, 3 or 4 times a day.
In one embodiment, the conjugate compound is administered to a human patient in accordance with the following dosage regimen: approximately 100 or approximately 125 mg, twice a day.
The dosage amounts described above can be applied to the conjugate (including the portion of PBD and the linker to the antibody) or to the effective amount of PBD compound provided, for example, the amount of compound that is releasable after cleavage of the linker.
For the prevention or treatment of a disease, the appropriate dosage of an ADC of the invention will depend on the type of disease that will be treated, as defined above, the severity and course of the disease, if the molecule is administered for preventive purposes. or therapeutic, previous therapy, the patient's clinical history and response to the antibody, and the criteria of the attending physician. The molecule it is properly administered to the patient once or during a series of treatments. Depending on the type and severity of the disease, about 1 mg / kg to 15 mg / kg (e.g., 0.1-20 mg / kg) of molecule is an initial candidate dosage for administration to the patient, either, for example, by a or more separate administrations, or by continuous infusion. A typical daily dosage could vary from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. An exemplary dosage of ADC which will be administered to a patient is in the range of from about 0.1 to about 10 mg / kg of the patient's weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of the symptoms of the disease occurs. An exemplary dosage regimen comprises a cycle of administration of an initial loading dose of about 4 mg / kg, followed by additional doses every week, two weeks, or three weeks of an ADC. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and tests.
Tra tamien t o The term "treatment", as used in the present document in the context of treating a condition, refers in general to treatment and therapy, either of a human or an animal (eg, in veterinary applications), where some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the degree of progress, a stop in the degree of progress, a regression of the condition, improvement of the condition, and cure of the condition. Treatment is also included as a prophylactic measure (ie, prophylaxis, prevention).
The term "therapeutically effective amount", as used herein, refers to the amount of an active compound, or a material, composition or dosage comprising an active compound, which is effective to produce some therapeutic effect. desired, proportional to a reasonable benefit / risk ratio, when administered according to a desired treatment regimen.
Similarly, the term "prophylactically effective amount", as used herein, refers to the amount of an active compound, or a material, composition or dosage comprising an active compound, which is effective for produce any desired prophylactic effect, proportional to a reasonable benefit / risk ratio, when administered according to a desired treatment regimen.
Preparation of Drug Conjugates Antibody-drug conjugates, as well as conjugates with other cell binding agents, can be prepared by various routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including the reaction of a group nucleophile of an antibody or a cell-binding agent with a drug-linker reagent. This method can be employed with various antibodies and cell binding agents to prepare the antibody-drug conjugates of the invention.
Nucleophilic groups on antibodies include, but are not limited to, side chain thiol groups, e.g., cysteine. The thiol groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups in linker portions such as those of the present invention. Certain antibodies have disulfides between reducible chains, that is, cysteine bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (Cleland reagent, dithiothreitol) or TCEP (tris (2-carboxyethyl) phosphine hydrochloride; Getz et al (1999) Anal.
Biochem. Vol 273: 73-80; Soltec Ventures, Beverly, MA). Each cysteine disulfide bridge will thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) which results in the conversion of an amine to a thiol.
The Subject / Patient The subject / patient may be an animal, mammal, a placental mammal, a marsupial (eg, kangaroo, Australian badger), a monotreme (eg, platypus), a rodent (eg, a guinea pig, a hamster, a rat). , a mouse), murine (for example, a mouse), a lagomorph (for example, a rabbit), avian (for example, a bird), canine (for example, a dog), feline (for example, a cat) , equine (for example, a horse), pig (for example, a pig), sheep (for example, a sheep), bovine (for example, a cow), a primate, ape (for example, an inferior ape or ape) superior), a lower ape (eg, marmoset, baboon), a superior ape (eg, gorilla, chimpanzee, orangutan, gibbon), or a human.
Additionally, the subject / patient can be in any of its forms of development, for example, a fetus. In a preferred embodiment, the subject / patient is a human.
In one embodiment, the patient is a population in which each patient has a tumor that has integrin anb on the surface of the cell.
Examples General Experimental Methods Optical rotations were measured in an ADP 220 polarimeter (Bellingham Stanlcy Ltd.) and the concentrations (c) are given in g / 100 i. The melting points were measured using a digital melting point (Electrothermal) apparatus. The IR spectra were recorded on a Spectrum 1000 FT IR spectrometer from Perkin-Elmer. The 1 H and 13 C NMR spectra were obtained at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported with respect to TEM (d = 0.0 ppm), and signals are designated s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) om (multiplet), with the coupling constants given in hertz (Hz). Mass spectroscopy (MS) data was collected using a Waters Micromass ZQ instrument coupled to a HPLC 2695 from Waters with a PDA 2996 from Waters. The Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source Temperature (° C), 100; Temperature of Desolvation (° C), 200; Cone flow rate (1 / h), 50; Desolvation flow rate (1 / h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Global QTOF Micromass in positive W mode using metal-coated borosilicate glass tips to introduce the samples in the instrument. Thin layer chromatography (TLC) was performed on silica gel aluminum plates (Merck 60, 254), and flash chromatography used silica gel (Merck 60, 230-400 mesh ASTM). Except for HOBt (NovaBiochem) and reagents supported on solid (Argonaut), all other chemicals and solvents were purchased from Sigma-Aldrich and used as supplied without further purification. Anhydrous solvents were prepared by distillation under an atmosphere of dry nitrogen in the presence of an appropriate drying agent, and stored on 4 A molecular sieves or sodium wire. Petroleum ether refers to the fraction that boils at 40-60 ° C.
General CL / MS conditions: Method 1 (default method, used unless otherwise indicated) HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0. 1%) and acetonitrile (B) (0.1% formic acid). Gradient: Initial composition of 5% B maintained for 1.0 min, then increase from 5% B to 95% B over a 3 min period. The composition was maintained for 0.1 min at 95% B, and then it was returned to 5% B in 0.03 minutes and remained there for 0.87 min. The total run time of the gradient is equal to 5 min.
Method 2 HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid). Gradient: initial composition of 5% of B maintained during 1.0 min, then increase of 5% of B to 95% of B during a period of 2.5 min. The composition was maintained for 0.5 min at 95% B, and then returned to 5% B in 0.1 minutes and remained there for 0.9 min. The total run time of the gradient is equal to 5 min.
For both methods Flow rate of 3.0 ml / min, 400 ml was fractionated by means of a Z-piece of zero dead volume that passes in the mass spectrometer. Wavelength detection interval: 220 to 400 nm. Function type: diode array (535 sweeps). Column: Phenomenex Onyx Monolithic C18 50 x 4.60 mm.
The phase flash purification conditions Inverse were the following: The Flash purification system (Varian 971-Fp) was run using a mobile phase of water (A) and acetonitrile (B). Gradient: initial composition of 5% B during 20 V.C. (Column Volume) then 5% B to 70% B within 60 V.C. The composition was maintained for 15 V.C. 95% of B, and subsequently returned to 5% of B in 5 V.C. and kept at 5% B for 10 V.C. The total run time of the gradient is equal to 120 V.C. Flow rate 6.0 ml / min. Wavelength detection interval: 254 nm. Column: Agilent AX1372-1 SF10-5.5gC8.
Preparative HPLC: Ultra-high performance reverse phase liquid chromatography (UPLC) was carried out on Phenomenex Gemini NX 5m C-18 columns of the following dimensions: 150 x 4.6 mm for analysis, and 150 x 21.20 m for work of preparation. All UPLC experiments were performed with gradient conditions. The eluents used were solvent A (H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid). The flow rates used were 1.0 ml / min for the analytical, and 20.0 ml / min for the preparative HPLC. Detection was performed at 254 and 280 nm.
Synthesis of the Intermediary 12 (a) 1 'r 3' Bis [2-methoxy-4- (methoxycarbonyl) phenoxy] propane (3) Diisopropyl azodicarboxylate (71.3 ml, 73.2 g, 362 mmol) was added dropwise over a period of 60 min to an excessive stirred solution of methyl vanillate 2 (60.0 g, 329 mmol) and Ph3P (129.4 g, 494 mmol) in anhydrous THF (800 ml) at 0-5 ° C (ice / acetone) under a nitrogen atmosphere. The reaction mixture was allowed to stir at 0-5 ° C for an additional 1 hour after which a solution of 1,3-propanediol (11.4 ml, 12.0 g, 158 mmol) in THF (12 ml) was added dropwise to the reaction mixture. drop for a period of 20 min. The reaction mixture was allowed to warm to room temperature and was stirred for 5 days. He The resulting white precipitate 3 was collected by vacuum filtration, washed with THF and dried in a vacuum desiccator to constant weight. Yield = 54.7 g (84% based on 1,3-propanediol). Satisfactory purity by LC / MS (3.20 min (ES +) m / z (relative intensity) 427 ([M + Na] +, 10); CH NMR (400 MHz, CDCl 3) d 7. 64 (dd, 2H, J = 1.8, 8.3 Hz), 7.54 (d, 2H, J = 1.8 Hz), 6. 93 (d, 2H, J = 8.5 Hz), 4.30 (t, 4H, J = 6.1 Hz), 3.90 (s, 6H), 3.89 (s, 6H), 2.40 (p, 2H, J = 6.0 Hz). (b) 1 ', 3' -Bis [2-methoxy-4- (methoxycarbonyl) -5-nitrophenoxypropane (4) Solid Cu (N03) 2-3H2O (81.5 g, 337.5 mmol) was slowly added to an excessive stirred suspension of bis-ester 3 (54.7 g, 135 mmol) in acetic anhydride (650 ml) at 0-5 ° C (ice /acetone). The reaction mixture was allowed to stir for 1 hour at 0-5 ° C and then allowed to warm to room temperature. A moderate exotherm (approx. 40-50 ° C), accompanied by the thickening of the mixture and the evolution of NO2 was observed in this stage. Additional acetic anhydride (300 ml) was added and the reaction mixture was allowed to stir for 16 hours at room temperature. The reaction mixture was poured onto ice (~ 1.51), stirred and allowed to return to room temperature. The resulting yellow precipitate was collected by vacuum filtration and dried in a Drying to give the desired compound bis-nitro 4 as a yellow solid. Yield = 66.7 g (100%). Satisfactory purity by LC / MS (3.25 min (ES +) m / z (relative intensity) 517 ([M + Na] +, 40); 1 H NMR (400 MHz, CDCI3) d 7.49 (s, 2H), 7.06 (s, 2H), 4.32 (t, 4H, J = 6.0 Hz), 3.95 (s, 6H), 3.90 (s, 6H), 2.45-2.40 (m, 2H). (c) 1 ', 3' -Bis (4-carboxy-2-methoxy-5-ni trofenoxy) propane (5) A suspension of methyl ester 4 (66.7 g, 135 mmol) in THF (700 mL) was treated with 1N NaOH (700 mL) and the reaction mixture was allowed to stir vigorously at room temperature. After 4 days of agitation, the suspension became a dark colored solution which was subjected to rotary evaporation under reduced pressure to remove the THF. The resulting aqueous residue was acidified to pH 1 with concentrated HCl and the colorless precipitate 5 was collected and dried thoroughly in a vacuum oven (50 ° C). Yield = 54.5 g (87%). Satisfactory purity by LC / MS (2.65 min (ES +) m / z (relative intensity) 489 ([M + Na] +, 30)); ¾ NMR (400 MHz, DMSO-d6) d 7. 62 (s, 2H), 7.30 (s, 2H), 4.29 (t, 4H, J = 6.0 Hz), 3.85 (s, 6H), 2.30-2.26 (m, 2H). (d) 1, 1 '- [[(Propane-1,3-diyl) dioxy] bis [(5-methoxy-2-nitro-1, 4-phenylene) carbonyl]] bis [(2S, 4R) -methyl] -4-hydroxypyrrolidine-2-carboxylate] (6) Oxalyl chloride (24.5 ml, 35.6 g, 281 moles) was added to a stirred suspension of nitrobenzoic acid 5 (43 g, 92.3 mmol) and DMF (6 ml) in anhydrous DCM (600 ml). After the initial effervescence, the reaction suspension became a solution and the mixture was allowed to stir at room temperature for 16 hours. The conversion to the acid chloride was confirmed by treating a sample of the reaction mixture with MeOH and the resulting bis-methyl ester was observed by LC / MS. The majority of the solvent was removed by evaporation under reduced pressure; The resulting concentrated solution was redissolved in a minimum amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was collected by filtration, washed with cold diethyl ether and dried for 1 hour in a vacuum oven at 40 ° C. The solid acid chloride was added in portions over a period of 25 min to a stirred suspension of (2S, 4R) -methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (38.1 g, 210 mmol) and TEA (64.5 ml, g, 463 mmole) in DCM (400 ml) at -40 ° C (dry ice / CH 3 CN).
Immediately, the reaction was complete as determined by LC / MS (2.47 min (ES +) m / z (relative intensity) 721 ([M + H] +, 100) The mixture was diluted with DCM (200 ml) and washed with 1 N HCl (300 mL), saturated NaHCOh (300 ml), saline (400 ml), dried (MgSO 4), filtered and the solvent was evaporated in vacuo to give the pure product 6 as an orange solid (66.7 g, 100%). [OI] 22D = -46.1 ° (c = 0.47, CHCl3); CH NMR (400 MHz, CDCl3) (rotamers) d 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28 (m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3. 46-3.38 (m, 2H), 3.02 (d, 2H, J = 11.1 Hz), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); 13 C NMR (100 MHz, CDCl 3) (rotamers) d 172.4, 166.7, 154.6, 148.4, 137.2, 127.0, 109.7, 108.2, 69. 7, 65.1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IR (ATR, CHCl3) 3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274, 1211, 1177, 1072, 1050, 1008, 871 cm1; MS (ES +) m / z (relative intensity) 721 ([M + H] +, 47), 388 (80); HRMS [M + H] + theoretical C31H36N4016 m / z 721.2199, (ES +) found m / z 721.2227. (e) 1, 1 '- [[(Propane-1, 3-diyl) dioxy] bis (llaS, 2R) -2- (hydroxy) -7-methoxy -1, 2, 3, 10, 11, hexahydro-5H-pyrrolo [2, 1 -c] [1,4] -benzodiazepin-5, 11-dione] (7) Method A: A solution of nitro-ester 6 (44 g, 61. 1 mmol) in MeOH (2.8 1) was added to freshly purchased Rancy® nickel (~ 50 g of a ~ 50% suspension in H2O) and anti-shock granules in a 3-nozzle, 3-round round bottom flask. The mixture was heated to reflux and then treated dropwise with a solution of hydrazine hydrate (21.6 ml, 22.2 g, 693 mmol) in MeOH (200 ral) moment in which vigorous effervescence was observed. When the addition was complete (~ 45 min) additional Rancy® nickel was carefully added until the effervescence ceased and the initial yellow color of the reaction mixture was removed. The mixture was refluxed for an additional 5 min at which time the reaction was considered complete by TLC (90:10 v / v CHCl 3 / MeOH) and LC / MS (2.12 min (ES +) m / z (relative intensity) 597 ([M + H] +, 100)). The reaction mixture was immediately hot filtered through a sintered funnel containing celite with vacuum suction. The filtrate was reduced in volume by vacuum evaporation at which time a colorless precipitate formed was collected by filtration and dried in a vacuum desiccator to provide 7 (31 g, 85%). [a] 27D = + 404 ° (c = 0. 10, DMF); 7H NMR (400 MHz, DMSO-dg) d 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H, J = 3.98 Hz, OH), 4.32- 4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62 (dd, 2H, J = 12.1, 3.60 Hz), 3.43 (dd, 2H, J = 12.0, 4.72 Hz), 2.67-2.57 (m, 2H), 2.26 (p, 2H, J = 5.90 Hz), 1.99- 1.89 (m, 2H); 13 C NMR (100 MHz, DMSO-ds) d 169.1, 164.0, 149. 9, 144.5, 129.8, 117.1, 111.3, 104.5, 54.8, 54.4, 53.1, 33. 5, 27.5; IR (ATR, pure) 3438, 1680, 1654, 1610, 1605, 1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038, 1018, 952, 870 cm1; MS (ES +) m / z (intensity relative) 619 ([M + Na] +, 10), 597. { [M + H] +, 52), 445 (12), 326 (11); HRMS [M + H] + theoretical C 29 H 32 N 4 O 10 / z 597.2191, (ES +) found m / z 597.2205.
Method B: A suspension of 10% Pd / C (7.5 g, 10% w / w) in DMF (40 ml) was added to a solution of nitro-ester 6 (75 g, 104 moles) in DMF (360 ml). ). The suspension was hydrogenated in a Parr hydrogenation apparatus for 8 hours. The progress of the reaction was monitored by LC / MS after the absorption of hydrogen had stopped. Solid Pd / C was removed by filtration and the filtrate was concentrated by rotary evaporation under vacuum (below 10 mbar) at 40 ° C to provide a dark oil containing traces of DMF and residual char. The residue was digested in EtOH (500 ml) at 40 ° C in a water bath (rotary evaporator bath) and the resulting suspension was filtered through celite and washed with ethanol (500 ml) to give a clear filtrate. Hydrazine hydrate (10 mL, 321 mmol) was added to the solution and the reaction mixture was heated to reflux. After 20 minutes the formation of a white precipitate was observed and the reflux was allowed to continue for an additional 30 minutes. The mixture was allowed to cool to room temperature and the precipitate was recovered by filtration, washed with diethyl ether (2: 1 volume of precipitate) and dried in a vacuum desiccator to provide 7 (50 g, 81%). The analytical data for method B: Identical to those obtained for Method A (optical rotation, 1H NMR, LC / MS and TLC). (f) 1, 1 '- [[(Propane-1, 3-diyl) dioxy] bis (llaS, 2R) -2- (tert-butmethylsilyloxy) -7-methoxy-1, 2, 3, 10, 11, 11a-hexahydro-5H-pyrrolo [2, 1-c] [1,4] benzodiazepin-5, 11-dione] (8) TBSC1 (27.6 g, 182.9 mmol) and imidazole (29.9 g, 438.8 mmol) were added to a cloudy solution of tetralactam 7 (21.8 g, 36.6 mmol) in anhydrous DMF (400 mL) at 0 ° C (ice / acetone). The mixture was allowed to stir under a nitrogen atmosphere for 3 hours after which time the reaction was considered complete as determined by LC / MS (3.90 min (ES +) m / z (relative intensity) 825 ([M + H] ] +, 100) The reaction mixture was poured onto ice (~ 1.75 1) and allowed to warm to room temperature with stirring The resulting white precipitate was collected by vacuum filtration, washed with H 2 O, diethyl ether and dried in the vacuum desiccator to provide 8 pure (30.1 g, 99%). [a] 23D = + 234 ° (c = 0.41, CHCl3); 3 H NMR (400 MHz, CDCl 3) d 8.65 (s, 2 H, N H), 7.44 (s, 2 H), 6.54 (s, 2 H), 4.50 (p, 2 H, J = 5.38 Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H), 3.73-3.63 (m, 4H), 2. 85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0. 86 (s, 18H), 0.08 (s, 12H); 13C NMR (100 MHz, CDC13) d 170. 4, 165.7, 151.4, 146.6, 129.7, 118.9, 112.8, 105.3, 69. 2, 65.4, 56.3, 55.7, 54.2, 35.2, 28.7, 25.7, 18.0, -4.82 and -4.86; IR (ATR, CHCl3) 3235, 2955, 2926, 2855, 1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099, 1033 enf1; MS (ES +) m / z (relative intensity) 825 ([M + H] +, 62), 721 (14), 440 (38); HRMS [M + H] + theoretical C4iH6oN4OioSi2 m / z 825.3921, (ES +) found m / z 825.3948. (g) i 1 '- [[(Propane-1, 3-diyl) dioxy] bis (llaS, 2R) -2- (tert-butyldimethylsilyloxy) -7-methoxy-10- ((2- (trimethylsilyl) ethoxy) methyl) -1, 2, 3, 10, 11, lla-hexahydro-5H-pyrrolo [2, 1-c] [1,4] -benzodiazepin-5, 11-dione] (9) A solution of n-BuLi (68.3 ml of a 1.6 M solution in hexane, 109 min) was added dropwise to a stirred suspension of tetralactam 8 (30.08 g, 36.4 mmol) in anhydrous THF (600 ml) at -30 °. C (dry ice / ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to stir at this temperature for 1 hour (now a reddish orange color) at which time a solution of SEMCI (19.3 ml, 18.2 g, 109 mmol) in anhydrous THF (120 ml) was added dropwise drop. The reaction mixture was allowed to slowly warm to room temperature and was stirred for 16 hours under a nitrogen atmosphere. The reaction was considered complete as determined by TLC (EtOAc) and LC / MS (4.77 min (ES +) m / z (relative intensity) 1085 ([M + H] +, 100).
The residue was removed by evaporation in vacuo and the resulting residue was dissolved in EtOAc (750 mL), washed with H 2 O (250 mL), saline (250 mL), dried (MgSO 4), filtered and evaporated in vacuo to provide the tetralactam protected with crude N10-SEM 9 as an oil (maxm 39.5 g, 100%). The product was carried through to the next step without purification. [a] 23D = + 163 ° (c = 0.41, CHCl3); 3 H NMR (400 MHz, CDCl 3) d 7.33 (s, 2 H), 7.22 (s, 2H), 5.47 (d, 2H, J = 9.98 Hz), 4.68 (d, 2H, J = 9.99 Hz), 4.57 (p, 2H, J = 5.77 Hz), 4.29-4.19 (m, 6H), 3.89 ( s, 6H), 3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J = 5.81 Hz), 2.03-1.90 (m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0. 01 (s, 18H); 13C NMR (100 MHz, CDC13) d 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9, 78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1, - 1.24, -4.73; IR (ATR, CHC13) 2951, 1685, 1640, 1606, 1517, 1462, 1433, 1360, 1247, 1127, 1065 crn-1; MS (ES +) m / z (Relative intensity) 1113 ([M + Na] +, 48), 1085 ([M + H] +, 100), 1009 (5), 813 (6); HRMS [M + H] + theoretical C53H88N4012SÍ4 m / z 1085.5548, (ES +) found m / z 1085.5542. (h) 1, 1 '- [[(Propane-1, 3-diyl) dioxy] bis (llaS, 2R) -2-hydroxy-7-methoxy-10- ((2- (trimethylsilyl) ethoxy) methyl) -1, 2, 3, 10, 11, lla-hexahydro-5H-pyrrolo [2, 1 -c] [1,4] -benzodiazepin-5, 11 -dione] (10) A solution of TBAF (150 ml of a 1.0 solution M in THF, 150 mmol) was added to a stirred solution of the crude bis-silyl ether 9 [84.0 g (max. 56.8 g), 52.4 immoles] in THF (800 ml) at room temperature. After stirring for 1 hour, analysis of the reaction mixture by TLC (95: 5 v / v CHCl 3 / MeOH) revealed the completion of the reaction. The THF was removed by evaporation under reduced pressure at room temperature and the resulting residue was dissolved in EtOAc (500 mL) and washed with NH 4 Cl (300 mL). The combined organic layers were washed with saline (60 mL), dried (MgSO4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (Gradient elution: 100% CHCl 3 at 96: 4 v / v CHCl 3 / MeOH) gave the pure tetralactam 10 as a white foam (36.0 g, 79%). LC / MS 3.33 min (ES +) m / z (relative intensity) 879. { [M + Na] +, 100), 857 ([M + H] +, 40); [a] 23D = + 202 ° (c = 0.34, CHCl3); ¾ NMR (400 MHz, CDC13) d 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J = 10.0 Hz), 4.72 (d, 2H, J = 10.0 Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J = 5.83 Hz), 4.20-4.16 (m, 2H), 3.91-3.85 (m, 8H), 3. 77-3.54 (m, 6H), 3.01 (br s, 2H, OH), 2.96-2.90 (m, 2H), 2. 38 (p, 2H, J = 5.77 Hz), 2.1 1-2.05 (m, 2H), 1.00-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDC13) d 169.5, 165. 9, 151.3, 147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69. 3, 67.2, 65.2, 56.5, 56.2, 54.1, 35.2, 29.1, 18.4, - I.23; IR (ATR, CHCl3) 2956, 1684, 1625, 1604, 1518, 1464, 1434, 1361, 1238, 1058, 1021 cm1; MS (ES +) m / z (relative intensity) 885. { [M + 29] +, 70), 857. { [M + H] +, 100), 711 (8), 448 (17); HRMS [M + H] + theoretical C41H60N4O12SÍ2 m / z 857.3819, (ES +) found m / z 857.3826. (i) 1, 1 '- [[(Propane-1, 3-diyl) dioxy] bis (llaS) -7-methoxy-2-oxo-10- ((2- (trimethylsilyl) ethoxy) methyl) -1, 2, 3, 10, - - - - 11-dione] (1D Diol 10 (25.6 g, 30 immoles, 1 eq.), NaOAc (6.9 g, 84 immoles, 2.8 eq.) And TEMPO (188 mg, 1.2 m ol, 0.04 eq.) Were dissolved in DCM (326 ml) under Ar. . This was cooled to -8 ° C (internal temperature) and TOCA (9.7 g, 42 mmol, 1.4 eq.) Was added in portions for 15 minutes. TLC (EtOAc) and LCMS [3.60 min. (ES +) m / z (relative intensity) 854.21 ([M + H] +, 40), (ES-) m / z (relative intensity) 887.07 ([M - H + CI], 10)] after 30 minutes indicated that the reaction was complete. DCM (200 ml) was added and the mixture was filtered through a pad of Celite before washing with a solution of saturated sodium hydrogencarbonate / sodium thiosulfate. (1: 1 v / v; 200 mi x 2). The organic layer was dried with MgSO 4, filtered and the solvent removed in vacuo to yield a yellow / orange sponge (25.4 g, 99%). LC / MS [3.60 min. (ES +) m / z (relative intensity) 854.21 ([M + H] +, 40); [a] 20D = + 291 ° (c = 0.26, CHCl3); X H NMR (400 MHz, CDCl 3) d 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J = 10.1 Hz), 4.75 (d, 2H, J = 10.1 Hz), 4.60 (dd, 2H, J = 9.85, 3.07 Hz), 4.31-4.18 (m, 6H), 3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd) , 2H, J = 19.2, 2.85 Hz), 2.76 (dd, 2H, J = 19.2, 9.90 Hz), 2. 42 (p, 2H, J = 5.77 Hz), 0.98-0.91 (m, 4H), 0.00 (s, 18H); 13 C NMR (100 MHz, CDC13) d 206.8, 168.8, 165.9, 151.8, 148.0, 133.9, 120.9, 111.6, 107.2, 78.2, 67.3, 65.6, 56.3, 54.9, 52.4, 37.4, 29.0, 18.4, -1.24; IR (ATR, CHC13) 2957, 1763, 1685, 1644, 1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 crrf1; MS (ES +) m / z (relative intensity) 881 ([M + 29] +, 38), 853 ([M + H] +, 100), 707 (8), 542 (12); HRMS [M + H] + theoretical C41H56N4O12SÍ2 m / z 853.3506, (ES +) found m / z 853.3502. (j) 1, 1 '- [[(Propane-1,3-diyl) dioxy] bis (1 laS) -7-methoxy-2- [[(trifluoromethyl) sulfonyl] oxy] -10- ((2- ( trimethylsilyl) ethoxy) methyl) -1, 10, 11, 1 la-tetrahydro-5H-pyrrolo [2, 1-c] [1,4] -benzodiazepin-5, 11-dione] (12) 2,6-Anhydrous lutidine (5.15 ml, 4.74 g, 44.2 mmol) was injected in one portion to a vigorously stirred solution of bis-ketone 11 (6.08 g, 7.1 mmol) in dry DCM (180 ml) at -45 ° C. (dry ice / acetonitrile) under a nitrogen atmosphere. Anhydrous triflic anhydride, taken from a freshly opened ampoule (7.2 ml, 12.08 g, 42.8 mmol), was injected rapidly dropwise, while the temperature was maintained at -40 ° C or below. The reaction mixture was allowed to stir at -45 ° C for 1 hour at which time TLC (50/50 v / v n-hexane / EtOAc) revealed the complete consumption of the starting material. The cold reaction mixture was immediately diluted with DCM (200 ml) and, with vigorous stirring, washed with water (1 x 100 ml), 5% solution of citric acid (1 x 200 ml) saturated NaHCO3 (200 ml) , saline solution (100 ml) and dried (MgSO 4). Filtration and evaporation of the solvent under reduced pressure gave the crude product which was purified by flash column chromatography (gradient elution: 90:10 v / v n-hexane / EtOAc at 70:30 v / v n-hexane / EtOAc) to give bis-enol 12 triflate as a yellow foam (5.5 g, 70%). LCMS 4.32 min (ES +) m / z (relative intensity) 1139 ([M + Na] +, 20); [a] 24D = + 271 ° (c = 0.18, CHCl3); 2 H NMR (400 MHz, CDCl 3) d 7.33 (s,2H), 7.26 (s, 2H), 7.14 (t, 2H, J = 1.97 Hz), 5.51 (d, 2H, J = 10.1 Hz), 4.76 (d, 2H, J = 10.1 Hz), 4.62 (dd, 2H, J = 11. 0, 3.69 Hz), 4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H, J = 16.3, 11.0, 2.36 Hz), 2.43 (p, 2H, J = 5.85 Hz), 1.23-0.92 (m, 4H), 0.02 (s, 18H); 13C NMR (100 MHz, CDC13) d 167.1, 162.7, 151.9, 148.0, 138.4, 133. 6, 120.2, 118.8, 111.9, 107.4, 78.6, 67.5, 65.6, 56.7, 56. 3, 30.8, 29.0, 18.4, -1.25; IR (ATR, CHCl3) 2958, 1690, 1646, 1605, 1517, 1456, 1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cirf1; MS (ES +) m / z (relative intensity) 1144 ([M + 28] +, 100), 1117 ([M + H] +, 48), 1041 (40), 578 (8); HRMS [M + H] + theory C43H54N40 i6Si2S2F6 m / z 1117. 2491, (ES +) found m / z 1117.2465.
Example 1 (a) Trifluoromethanesulfonate of (S) -8- (3- (((S) -2 (4-aminophenyl) -7-methoxy-5,1-l-dioxo-10- ((2 (trimethylsilyl) ethoxy) methyl) -5,10,11, lla-tetrahydro-lH-benzo [e] pyrr lo [1,2- a] [1,4] diazepin-8-yl) oxy) propoxy) - 7 -methoxy -5,1-dioxo-10- ((2 - (trimethylsilyl) ethoxy) methyl) 5,10,11, lla-tetrahydro-lH-benzo [e] pyrrolo [1, 2- a] [1,4] diazepin-2-yl (13) Pd (PPh3) 4 (116.9 mg, 0.101 mmol) was added to a stirred mixture of bis-enol triflate 12 (5.65 g, 5.06 mmol), 4-aminophenylboronic acid pinacol ester (1 g, 4.56 mmol), Na2CO3 (2.46 g, 23.2 mmol), MeOH (37 mL), toluene (74 mL) and water (37 mL). The reaction mixture was allowed to stir at 30 ° C under a nitrogen atmosphere for 24 hours after which all the boronic ester was consumed. The reaction mixture was then evaporated until the residue was taken up in EtOAc (150 mL) and washed with H2O (2 x 100 mL), saline (150 mL), dried (MgSO4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 80:20 v / v Hexane / EtOAc at 60:40 v / v Hexane / EtOAc) provided the product 13 as a yellowish foam (2.4 g, 45%). LC / MS 4.02 min (ES +) m / z (relative intensity) 1060.21 ([M + H] +, 100); 1H-NMR: (CDCl3, 400 MHz) d 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H), 7.24 (d, 2H, J = 8.5 Hz), 7.15 (t, 1H, J = 2.0 Hz), 6. 66 (d, 2H, J = 8.5 Hz), 5.52 (d, 2H, J = 10.0 Hz), 4.77 (d, 1H, J = 10.0 Hz), 4.76 (d, 1H, J = 10.0 Hz), 4.62 (dd, 1H, J = 3.7, 11.0 Hz), 4.58 (dd, 1H, J = 3.4, 10.6 Hz), 4. 29 (t, 4H, J = 5.6 Hz), 4.00-3.85 (m, 8H), 3.80 - 3.60 (m, 4H), 3.16 (ddd, 1H, J = 2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H, J = 2.2, 10.5, 16.1 Hz), 2.43 (p, 2H, J = 5.9 Hz), 1.1 -0.9 (m, 4H), 0.2 (s, 18H). 13 C-NMR: (CDC13, 100 MHz) d 169.8, 168.3, 164.0, 162.7, 153.3, 152.6, 149.28, 149.0, 147. 6, 139.6, 134.8, 134.5, 127.9, 127.5, 125.1, 123.21, 121. 5, 120.5, 120.1, 116.4, 113.2, 108.7, 79.8, 79.6, 68.7, 68. 5, 67.0, 66.8, 58.8, 58.0, 57.6, 32.8, 32.0, 30.3, 19.7, 0.25. (b) (S) -2- (4-Aminophenyl) -8- (3- (((S) -2-cyclopropyl-7-methoxy-5,1-dioxo-10- ((2- (trimethylsilyl) ethoxy ) methyl) -5, 10, 11, lla-tetrahydro-lH-benzo [e Jpirrolo [1, 2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7-methoxy-10- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-benzo [e] pyrrolo [1, 2-a] [1,4] diazepine-5, 11 (10H, llaH) -dione (14) Trifenilarsine (0.24 g, 0.8 mmol), silver oxide (I) (1.02 g, 4.4 mmol), cyclopropylboronic acid (0.47 g, 5.5 mmol) and starting material 13 (1.15 g, 1.1 mmol) were dissolved in dioxane (30 g). mi) under an argon atmosphere. Triassic potassium phosphate (2.8 g, 13.2 mmol) was ground using a pestle and mortar and added quickly to the reaction mixture. The reaction mixture was evacuated and purged with argon 3 times and heated to 71 ° C. Bis (benzonitrile chloride) palladium (II) (84 mg, 0.22 mmol) was added and the reaction vessel was evacuated and purged with argon 3 times. After 10 minutes a small sample was taken for analysis by TLC (80:20 v / v ethyl acetate / hexane) and LC / MS. After 30 minutes, the reaction was complete (the LC / MS analysis indicated complete consumption of the starting material) and the reaction was filtered through celite and the filter pad was washed with ethyl acetate (400 ml) . The filtrate was washed with water (2 x 200 i) and saline (2 x 200 ml). The organic layer was dried with MgSO 4, filtered and the solvent was removed in vacuo.
Purification by silica gel column chromatography (30:70 v / v Hexane / ethyl acetate) gave product 14 as an orange / yellow solid (0.66 g, 63%). Method 1, LCMS (3.85 min (ES +) m / z (relative intensity) 952.17 ([M + H] +, 100) XH NMR (400 MHz, CDCl3) d 7.36 (d, 2H, J = 8.4 Hz ), 7.30 (s, 1H), 7.25 - 7.19 (m, 4H), 6.68 (s, 1H), 6.62 (d, 2H, J = 8. 4 Hz), 5.49 (dd, 2H, J = 5.6, 10.0 Hz), 4.73 (app.t, 2H, J = 10.8 Hz), 4.54 (dd, 1H, J = 3.2, 10.4 Hz), 4.40 (dd, 1H, J = 3.2, 10.4 Hz), 4.29 - 4.23 (m, 4H), 3.91 - 3.85 (m, 7H), 3.80 - 3.71 (m, 2H), 3.70 -3.61 (m, 2H), 3.38 - 3.32 (m, 1H), 3.12 - 3.01 (m, 1H), 2.50 - 2.69 (m, 1H), 2.40 (q, 2H, J = 5.6 Hz), 1.50 - 1.43 (m, 1H), 0.99 - 0.71 (m, 6H), 0. 54-59 (m, 2H), 0. 00 (s, 18H) ppm. (c) (S) -2- (4-Aminophenyl) -8- (3 - (((S) -2-cyclopropyl-7-methoxy-5-oxo-5, lla-dihydro-lH- benzo [ejirrolo [1, 2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7-methoxy-lH-benzo [ejirrolo [1,2-a] [1,4] diazepin-5 (llaH) -one (fifteen) Dilactam SEM 14 (0.66 g, 0.69 mmol) was dissolved in THF (23 mL) and cooled to -78 ° C under an argon atmosphere. Super-Hydride® solution (1.7 ml, 1 M in THF) was added dropwise during 5 minutes while temperature was monitored. After 20 minutes, a small sample was taken and washed with water for LC / MS analysis. Water (50 ml) was added and the cold bath was removed. The organic layer was extracted and washed with saline (60 i). The combined aqueous layers were washed with CH2Cl2 / MeOH (90/10 v / v) (2 x 50 mL). The combined organic layers were dried with MgSO 4, filtered and the solvent removed in vacuo. The crude product was dissolved in MeOH (48 mL), CH2Cl2 (18 mL) and water (6 mL) and sufficient silica gel was added to give a slurry. After 5 days of stirring, the suspension was filtered through a sintered funnel and washed with CH2Cl2 / MeOH (9: 1) (~200 ml) until the product was no longer eluted. The organic layer was washed with saline (2 x 70 mL), dried with MgSO 4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (100% CHCI3 at 96/4 v / v CHCl 3 / MeOH) gave the product as a yellow solid (302 g, 66%). Method 1, LCMS (2.42 min (ES +) m / z (relative intensity) 660.74 ([M + H] 30) CH NMR (400 MHz, CDCl 3) d 7. 86 (d, 1H, J = 3.6 Hz), 7.78 (d, 1H, J = 3.6 Hz), 7.58 -7.44 (m, 3H), 7.34 - 7.20 (m, 3H), 6.88 - 6.66 (m, 4H) , 4.35 - 4.15 (m, 6H), 3.95 - 3.75 (m, 7H), 3.39 - 3.22 (m, 1H), 3.14 - 3.04 (m, 1H), 2.93 - 2.85 (m, 1H), 2.46 -2.36 ( m, 2H), 1.49-1.41 (m, 1H), 0.80-0.72 (m, 2H), 0.58-0.51 (app.s, 2H) ppm. (d) ((2S) -l- (((2S) -1- ((4- (8- (3- ((2-cyclopropyl-7-methoxy-5-oxo-5, lla-dihydro-lH- benzo [ejpirrolo [1, 2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7-methoxy-5-oxo-5, 11a-dihydro-lH-benzo [ejirrolo [1,2- allyl [1, 4] diazepin-2-yl) phenyl) amino) -l-oxopropan-2-yl) amino) -3-methyl-l-oxobutan-2-yl) carbamate (16) In a degassed round bottom flask filled with argon, HO-Ala-Val-alloc (149.6 mg, 0.549 mmol) and EEDQ (135.8 mg, 0.549 mmol) were dissolved in a 9: 1 mixture of dry CH2Cl2 / MeOH (5 mL). ). The flask was wrapped in aluminum foil and the reaction mixture was allowed to stir at room temperature for 1 hour before the starting material 15 was added (302 mg, 0.457 mmol). The reaction mixture was allowed to stir for an additional 40 hours at room temperature before the volatiles were removed by rotary evaporation under reduced pressure (the reaction was followed by LC / MS, RT starting material 2.32 min, (ES + 660.29 ([M + H] +, 100)).
The crude product was purified directly by silica gel column chromatography (100% CHCl 3 at 90/10 v / v CHCl 3 / MeOH) to give the pure product (16) in 42% yield (174 mg). Method 2 CL / MS (2.70 min (ES +) m / z (relative intensity) 914.73 ([M + H] +, 60), 660. 43 (60), 184.31 (100)). (e) (2S) -2-amino-N- ((2S) -1- ((4- (8- (3- ((2-cyclopropyl-7-methoxy-5-oxo-5, lla-dihydro- lH-benzo [ejpirrolo [1, 2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7-methoxy-5-oxo-5, lla-dihydro-lH-benzo [e] pyrr [1, 2-a] [1, 4] diazepin-2-yl) phenyl) amino) -l-oxopropan-2-yl) -3-me t i lbutylnamide (17) The starting material 16 (170 mg, 0.185 mmol) was dissolved in dry CH 2 Cl 2 (5 i) in a round bottom flask filled with argon, before pyrrolidine (41 ml, 0.21 mmol) was added. The flask was purged / refilled three times with argon before Pd (PPh3) 4 (14 mg, 0.084 mmol) was added and the washing operation was repeated. After 1 hour, the complete consumption of the starting material was observed (the reaction was followed by LC / MS) and Et20 (50 ml) was added to the reaction mixture which was allowed to stir until all the product had crashed out of the solution. The solid was filtered through a sintered funnel and washed twice with Et30 (2 x 25 mL). The collecting flask is replaced and the isolated solid was dissolved in CHCl3 (100 ml or until all the product had passed through the sintered funnel). The volatiles were then removed by rotary evaporation under reduced pressure to provide the crude product 17 which was used directly in the next step (168 mg). Method 2 LC / MS (2.70 min (ES +) m / z (relative intensity) 830.27 ([M + H] +, 50), 660.13 (80), 171.15 (100)). (f) N- ((R-1 - (((S) -l- ((4- ((S) -8- (3- (((S) -2-cyclopropyl-7-methoxy-5-oxo -5, lla-dihydro-lH-benzo [e] pyrr lo [1, 2-a] [1, 4] diazepin-8-yl) oxy) propoxy) -7-methoxy-5-oxo-5, dihydro-lH-benzo [e Jpirrolo [1, 2-a] [1,4] diazepin-2-yl) phenyl) amino) -l-oxopropan-2-yl) amino) -3-methyl-l-oxobutan- 2-yl) -1- (3- (2, 5-dioxo-2, 5-dihydro-lH-pyrrol-1-yl) propanamide) -3, b, 9, 12, 15, 18, 21, 24- octaoxaheptacosan-27 -amide (18) The starting material 17 (154 mg, 0.185 mmol) and EDC1.HC1 (110 mg, 0.185 mmol) was solubilized in dry CH2Cl2 (5 mL) in a round-bottomed flask purged and filled with argon. The mixture was allowed to stir at room temperature for 1 hour before PEGg-maleimide (35.6 mg, 0.185 mmol) was added and the reaction mixture was stirred for an additional 16 hours (or until the reaction was complete, monitored by LC / EM). The reaction solution was diluted with CH2C12 (50 i) and the organics were washed with H2O (50 ml) and saline (50 i) before being dried with MgSC > 4, filtered and the solvent was removed by rotary evaporation under reduced pressure to provide the crude product. Purification on silica gel column chromatography (100% CHCl 3 at 85/15 v / v CHCl 3 / MeOH) gave the desired product (135 mg), however, remaining traces of unreacted PEGs-maleimide were observed ( by LC / MS, 2.21 min, method 2). Automated reverse phase silica gel chromatography (H2O / CH3CN) (see general information for conditions) successfully eliminated the impurity by providing the pure final product (18.37 mg of pure product from 110 mg, 33%). Overall yield = 17%. Method 2 LC / MS (2.58 min (ES +) m / z (relative intensity) 1404.03 ([M + H] +, 20), 702.63 (100)). X H NMR (400 MHz, CDCl 3) d 7.91 (t, J = 3.5 Hz, 1 H), 7.80 (d, J = 4.0 Hz, 1 H), 7.75 (d, J = 8.8 Hz, 1 H), 7.69 (d, J = 8.7 Hz, 1H), 7.54 - 7.50 (m, 2H), 7.45 (s, 1H), 7.39 - 7.31 (m, 2H), 6.87 (d, J = 10.5 Hz, 2H), 6.76 (s, 1H) , 6.72-6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67 - 3.58 (m, 34H) , 3.54 (m, 2H), 3.42 (dd, J = 10.2, 5.2 Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J = 16.1, 4.1 Hz, 1H), 2.62-2.49 (m , 4H), 2.48-2.39 (, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79 (dd, J = 9.2, 5.3 Hz, 2H), 0.57 (dd, J = 9.2, 5.3 Hz, 2H), no observed NH.
Example 2 (a) Acid (R) -2- ((R) -2- ((((9H-Fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido propanoic acid (20b) HO-Ala-Val-H 20a (350 mg, 1.86 mmol) and Na 2 CO 3 (493 mg, 4.65 mmol) were dissolved in distilled H 2 O (15 mL) and the mixture was cooled to 0 ° C before dioxane was added (15 mL). mi) (partial precipitation of the amino acid salt occurred). A solution of Fmoc-Cl (504 mg, 1.95 mmol) in dioxane (15 mL) was added dropwise with vigorous stirring for 10 minutes. The resulting mixture was stirred at 0 ° C for 2 hours before the ice bath was removed and stirring was maintained for 16 hours. The solvent was removed by rotary evaporation under reduced pressure and the residue was dissolved in water (150 ml). The pH was adjusted from 9 to 2 with 1N HCl and, subsequently, the aqueous layer was extracted with EtOAc (3x100 i). The combined organics were washed with saline (100 ml), dried with MgSO 4, filtered and the volatiles were removed by rotary evaporation under reduced pressure to provide pure HO-Ala-Val-Fmoc 20b (746 mg, 97% yield). ). LCMS 2.85 min (ES +) m / z (relative intensity) 410.60; 1 H-NMR (400 MHz, CDCl 3) d 7.79 (d, J = 1.77 Hz, 2H), 7.60 (d, J = 7.77 Hz, 2H), 7.43 (d, J = 7.5 Hz, 2H), 7.34 (d, J = 7.5 Hz, 2H), 6.30 (bs, 1H), 5.30 (bs, 1H), 4.71-7.56 (m, 1H), 4.54-4.36 (m, 2H), 4.08-3.91 (m, 1H), 2.21-2.07 (m, 1H), 1.50 (d, J = 7.1 Hz, 3H) , 1.06-0.90 (m, 6H). (b) ((S) -3-methyl-l-oxo-l- (((S) -1-oxo-l- ((4- (4, 4, 5, 5-tetramethyl-l, 3, 2 dioxaborolan-2-yl) phenyl) amino) propan-2-yl) amino) butan-2-yl) carbamate of (9H-fluoren-9-yl) methyl (twenty) Pinacol ester of 4-aminophenylboronic acid (146.9 mg, 0.67 mmol) was added to a solution of HO-Ala-Val-Fmoc 20b (330 mg, 0.8 mmol), DCC (166 mg, 0.8 mmol) and DMAP (5 mg). mg, cat.) in dry DCM (8 ml) was previously stirred for 30 minutes at room temperature in a flask purged with argon. The reaction mixture was then allowed to stir at room temperature overnight. The reaction was followed by LCMS and TLC. The reaction mixture was diluted with CH2Cl2, and the organics were washed with H2O and saline before being dried with MgSO4, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was charged dry on a silica gel chromatography column (Hexane / EtOAc, 6: 4) and the pure product was isolated as a white solid in 88% yield (360 mg). (c) Trifluoromethanesulfonate 8- (3- ((2- (4- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) - 3- methylbutanamido) propanamido) phenyl) -7-methoxy-5, 11-dioxo-10- ((2 - (trimethylsilyl) ethoxy) methyl) -5, 10, 11, lla-tetrahydro-lH-benzo [ejirrolo [1, 2] α] [1,4] diazepin-8-yl) oxy) propoxy) -7-methoxy-5,11-dioxo-l 0 - ((2- (trimethylsilyl) ethoxy) methyl) -5, 10, 11, lla-tetrahydro-lH-benzo [ejpirrolo [1,2-a] [1,4] diazepin-2-yl (2D Bis-triflate 12 (2.03 g, 1.81 mmol), boronic pinacol ester (1 g, 1.63 mmol) and Na 2 CO 3 (881 mg, 8.31 mmol) were dissolved in a toluene / eOH / H 2 O mixture, 2: 1: 1 ( 40 mi). The reaction flask was purged and filled with argon three times before tetrakis (triphenylphosphine) palladium (0) (41 mg, 0.035 mmol) was added and the reaction mixture was heated at 30 ° C overnight. The solvents were removed under reduced pressure and the residue was taken up in H20 (100 ml) and extracted with EtOAc. (3 x 100 mi). The combined organics were washed with saline (100 ml), dried with MgSO 4, filtered and the volatiles were removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography (Hexane / EtOAc, 8: 2 to 25:75) to give pure 21 in 33% yield (885 mg). LC / MS 3.85 min (ES +) m / z (relative intensity) 1452.90; 1 H NMR (400 MHz, CDCl 3) d 7.78 - 7.16 (m, 17 H), 7.13 (s, 1 H), 6.51 - 6.24 (m, 1H), 5.51 (dd, J = 10.0, 5.1 Hz, 2H), 5.36 - 5.11 (m, 1H), 4.74 (dd, J = 10.1, 4.4 Hz, 2H), 4.70-4.53 (m, 2H), 4. 47 (d, J = 6.4 Hz, 1H), 4.37 (d, J = 7.2 Hz, 1H), 4.27 (m, 4H), 4.20 - 4.14 (m, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3. 77 (ddd, J = 16.7, 9.0, 6.4 Hz, 3H), 3.71 - 3.61 (m, 2H), 3. 24 - 2.91 (m, 3H), 2.55 - 2.33 (m, 2H), 2.22 - 2.07 (m, 1H), 1.52 - 1.37 (m, 3H), 1.04 - 0.86 (m, 10H), 0.00 (s) , 18H). (d) ((2S) -1 (((2S) -1- ((4- (8- (3- ((2-cyclopropyl-1-methoxy-5, 11-dioxo-l 0 - ((2- (trimethylsilyl) ethoxy) methyl) -5, 10, 11, lla-tetrahydro-lH-benzo [e] pyrr lo [1, 2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7 -methoxy-5, 11-dioxo-10- ((2- (trimethylsilyl) ethoxy) ethyl) -5, 10,11, lla-tetrahydro-lH-benzo [ejirrolo [1, 2-a] [1, 4] diazepin-2-yl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-l-oxobutan-2-yl) carbamate of (9H-fluoren-9-yl) methyl (22) Trifenilarsine (42 mg, 0.137 mmol) was added to a mixture of PBD-triflate 21 (250 mg, 0.172 mmol), cyclopropylboronic acid (73.9 g, 0.86 mmol), silver oxide (159 mg, 0.688 mmol) and potassium phosphate tribasic (438 mg, 2.06 mmol) in dry dioxane (10 ml) under an argon atmosphere. The reaction was purged with argon 3 times and bis (benzonitrile) palladium (II) chloride (13.2 mg, 0.034 mmol) was added. The reaction was purged with argon 3 more times before being heated to 75 C and stirred for 10 minutes. The reaction mixture was filtered through a Celite pad which was subsequently rinsed with ethyl acetate. The solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel, 1% methanol / chloroform). The pure fractions were collected and combined, and the excess eluent was removed by rotary evaporation under reduced pressure to provide the desired product 22 (132 mg, 50% yield). LC / MS 3.83 min (ES +) m / z (relative intensity) 1345.91; CH NMR (400 MHz, CDCl3) d 7.88-1.14 (m, 17H), 6.69 (s, 1H), 6.45-6.25 (m, 1H), 5.57-5.41 (m, 2H), 5.34-5.14 (m, 1H) ), 4.78 - 4.67 (, 2H), 4.62 - 4.55 (m, 1H), 4.50 - 4.45 (m, 2H), 4.51 - 4.44 (m, 1H), 4.31 -4.21 (, 4H), 4.16 (m, 1H) ), 3.92 (s, 3H), 3.86 (s, 3H), 3.82 - 3.71 (m, 2H), 3.66 (m, 3H), 3.40 - 3.28 (m, 1H), 3. 07 (m, 1H), 2.70 - 2.57 (m, 1H), 2.47 - 2.36 (m, 2H), 2. 15 (m, 1H), 1 .51-1.40 (m, 3H), 1.03-0.87 (m, 11H), 0. 77 - 0.71 (m, 2H), 0.60 - 0.54 (m, 2H), 0.00 (t, J = 3.0 Hz, 18H). (e) ((2S) -1 - (((2S) -l - ((4- (8- (3- ((2-cyclopropyl-7-methoxy-5-oxo-5, 1-dihydro-1H -benzo [e] pyrr [1, 2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7-methoxy-5 -oxo-5, 11a-dihydro-lH-benzo [e] pyrr it [1, 2-a] [1,4] diazepin-2-yl) phenyl) amino) -l -oxopropan-2-yl) amino) -3-methyl-l-oxobutan-2-yl) carbamates of (9H-fluoren-9-yl) methyl (23) A solution of Super-Hydride® (0.5 mL, 1M in THF) was added dropwise to a solution of dilactam SEM 22 (265 mg g, 0.19 mmol) in THF (10 mL) at -78 ° C under an atmosphere of argon. The addition was completed in 5 minutes in order to keep the internal temperature of the reaction mixture constant. After 20 minutes, an aliquot was quenched with water for LC / MS analysis, which revealed that the reaction was completed. Water (20 i) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with EtOAc (3 x 30 mL) and the combined organics were washed with saline (50 mL), dried with MgSO 4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dissolved in MeOH (12 i), CH 2 Cl 2 (6 i), water (2 ml) and sufficient silica gel to form a suspension under thick agitation. After 5 days, the suspension was filtered through a sintered funnel and washed with CH2Cl2 / MeOH (9: 1) (200 ml) until the elution of the product was complete. The organic layer was washed with saline (2 x 70 i), dried with MgSO 4, filtered and the solvent was removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (100% CHCI3 to 96% CHCl3 / 4 MeOH) provided product 23 as a yellow solid (162 mg, 78%). LC / MS 3.02 min (ES +) m / z (relative intensity) 1052.37. (f) (2S) -2-amino-N- ((2S) -1 - ((4- (8- (3- ((2-cyclopropyl-7-methoxy-5-oxo-5,1-dihydro) - 1 H -benzo [e] pyrro [1,2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7-methoxy-5-oxo-5, lla-dihydro-lH-benzo [ e] pyrr lo [1, 2 -a] [1, 4] diazepin-2-yl) phenyl) amino) -1-oxopropan-2-yl) -3-methylbutynamide (17) Excess piperidine (0.2 ml, 2 min) was added to a solution of SEM-dilactam 23 (76 mg, 0.073 mmol) in DMF (1 mi). The mixture was allowed to stir at room temperature for 20 min, at which time the reaction was completed (as monitored by LC / MS). The reaction mixture was diluted with CH2Cl2 (75 mL) and the organic phase was washed with H2O (3x75 mL) until complete removal of piperidine. The organic phase was dried over MgSO 4, filtered and the excess solvent was removed by rotary evaporation under reduced pressure to provide the crude product 17 which was used as such in the next step. LC / MS 2.32 min (ES +) m / z (relative intensity) 830.00. (g) N ((2S) -1 - (((2S) -1 - ((4- (8- (3- ((2-cyclopropyl-7-methoxy-5-oxo-5, lla-dihydro-lH -benzo [e] pyrr lo [1, 2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7-me t-oxy-5 -oxo-5, 11a-dihydro-lH-benzo [ e] pyrr [1, 2-a] [1,4] diazepin-2-yl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1 - oxobutan-2-yl) -1 - (3- (2, 5-dioxo-2, 5-dihydro-lH-pyrrol-l-yl) propanamido) -3, b, 9, 12, 15, 18, 21, 24-octaoxaheptacosan-27-amide (18) EDCI Hydrochloride (14 mg, 0.0732 mmol) was added to a suspension of Maleimide-PEG8-acid (43.4 mg, 0.0732 mmol) in dry CH2Cl2 (5 mL) under an argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 17 (60.7 mg, 0.0732 mmol) was added. Agitation was maintained until the reaction was completed (usually 5 hours). The reaction was diluted with CH2C12 and the organic phase was washed with H2O and saline before being dried over MgSO4, filtered and the excess solvent was removed by rotary evaporation under reduced pressure. The product was carefully purified by silica gel chromatography (slow elution starting with 100% CHCl3 to 9: 1 CHCls / MeOH), followed by reverse phase chromatography to remove maleimide-PEGs-unreacted acid. The product 18 was isolated in 17.6% (21.8 mg). LC / MS 2.57 min (ES +) m / z (relative intensity) 1405.30; (400 MHz, CDCl3) d 7.91 (t, J = 3.5 Hz, 1H), 7.80 (d, J = 4.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (s, 1H), 7. 39 - 7.31 (m, 2H), 6.87 (d, J = 10.5 Hz, 2H), 6.76 (s, 1H), 6.72-6.68 (m, 2H), 4.74 -4.62 (m, 1H), 4.45 - 4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67 3.58 (m, 34H), 3. 54 (m, 2H), 3.42 (dd, J = 10.2, 5.2 Hz, 2H), 3.16 - 3.07 (m, 1H), 2.92 (dd, J = 16.1, 4.1 Hz, 1H), 2.62 - 2.49 (m, 4H), 2.48-2.39 (m, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79 (dd, J = 9.2, 5.3 Hz, 2H), 0.57 (dd, J = 9.2, 5.3 Hz, 2H), no NH was observed.
Example 3 (a) Trifluoromethanesulfonate of (S) -7-methoxy-8- (3- (((S) -7-methoxy-2- (4- (4-methyl-piperazin-1-yl) phenyl) -5, 11- dioxo-10- ((2- (trimethylsilyl) ethoxy) methyl) -5, 10, 11, 11a-tetrahydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy ) -5, 11-dioxo-10- ((2- (trimethylsilyl) ethoxy) methyl) -5, 10, 11, lla-tetrahydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-2 -ilo (24) Pd (PPh3) 4 (20.6 mg, 0.018 mmol) was added to a stirred mixture of bis-enol triflate 12 (500 mg, 0.44 mmol), boronic ester of N-methyl piperazine (100 mg, 0.4 mmol), Na2CO3 ( 218 mg, 2.05 mmol), MeOH (2.5 mL), toluene (5 mL) and water (2.5 mL). The reaction mixture was allowed to stir at 30 ° C under a nitrogen atmosphere for 24 hours after which all the boronic ester was consumed. The reaction mixture was then evaporated until the residue was taken up in EtOAc (100 ml) and washed with H2O (2 x 50 ml)., saline (50 ml), dried (MgSO4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 80:20 v / v Hexane / EtOAc at 60:40 v / v Hexane / EtOAc) afforded product 24 as a yellowish foam (122.6 mg, 25%). LC / MS 3.15 min (ES +) m / z (relative intensity) 1144 ([M + H] +, 20%). (b) ((S) -1 - (((S) -1 - ((4- ((S) -7-methoxy -8- (3- (((S) - 7-methoxy-2- (4- (4-methylpiperazin-1-yl) phenyl) -5,1-dioxo-10- ((2- (trimethylsilyl) ethoxy) methyl) -5, 10, 11, tetra-tetrahydro -lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -5, 11-dioxo-10- ((2- (trimethylsilyl) ethoxy) methyl) -5, 10 , 11, 11a-tetrahydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-2-yl) phenyl) amino) -l-oxopropan-2-yl) amino) -3-methyl-l- oxobutan-2-yl) carbamate (9H-fluoren-9-yl) methyl (25) PBD-triflate 24 (359 mg, 0.314 mmol), boronic pinacol ester 20 (250 mg, 0.408 mmol) and triethylamine (0.35 mL, 2.51 mmol) were dissolved in a toluene / MeOH / PO mixture, 2: 1: 1 (3 mL). The microwave vessel was purged and filled with argon three times before tetrakis (triphenylphosphine) palladium (0) (21.7 mg, 0.018 mmol) was added and the reaction mixture was placed in the microwave at 80 ° C for 10 minutes. . Subsequently, CH2Cl2 (100 mL) was added and the organics were washed with water (2 x 50 mL) and saline (50 mL) before being dried with MgSO4, filtered and the volatiles were removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography (CHCl3 / MeOH, 100% to 9: 1) to give pure (200 mg, 43% yield). LC / MS 3.27 min (ES +) m / z (relative intensity) 1478 ([M + H] +, 100%). (c) ((S) 1 - (((S) 1 - ((4- ((S) -7-methoxy-8- (3- (((S) -7-methoxy-2- (4- ( 4-methylpiperazin-l -yl) phenyl) -5 -oxo-5, lia- dihydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -5-oxo-5, lla-dihydro-lH-pyrrolo [2, 1-c] [1 , 4] benzodiazepin-2-yl) phenyl) amino) -l-oxopropan-2-yl) amino) -3-methyl-l-oxobutan-2-yl) carbamame of (9H-fluoren-9-yl) methyl (26) A solution of Super-Hydride® (0.34 mL, 1M in THF) was added dropwise to a solution of SEM-dilactam 25 (200 mg, 0.135 mol) in THF (5 mL) at -78 ° C under an atmosphere of argon. The addition was completed in 5 minutes in order to keep the internal temperature of the reaction mixture constant. After 20 minutes, an aliquot was quenched with water for LC / MS analysis, which revealed that the reaction was completed. Water (20 ml) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with EtOAc (3 x 30 mL) and the combined organics were washed with saline (50 mL), dried with MgSO 4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dissolved in MeOH (6 mL), CH2Cl2 (3 mL), water (100 mL) and sufficient silica gel to form a suspension under thick agitation. After 5 days, the suspension was filtered through a sintered funnel and washed with CH2Cl2 / MeOH (9: 1) (100 ml) until the elution of the product was complete. The organic layer was washed with saline (2 x 50 ml), dried with MgSO 4, filtered and the The solvent was removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (100% CHCl 3 to 96% CHCl 3/4% MeOH) provided product 26 as a yellow solid (100 mg, 63%). LC / MS 2.67 min (ES +) m / z (relative intensity) 1186 ([M + H] +, 5%). (d) (S) -2-amino-N- ((S) -1- ((4- ((R) -7-methoxy-8- (3- (((R) -7-methoxy-2- (4- (4-methylpiperazin-l-yl) phenyl) -5-oxo-5, lla-dihydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -5-oxo-5, lla-dihydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-2-yl) phenyl) amino) -l-oxopropan-2-yl) -3-met i Ibut anamida (27) Excess piperidine (0.1 ml, 1 mmol) was added to a solution of PBD 26 (36.4 mg, 0.03 mmol) in DMF (0.9 mL). The mixture was allowed to stir at room temperature for 20 min, at which time the reaction was complete (as monitored by LC / MS). The reaction mixture was diluted with CH2Cl2 (50 mL) and the organic phase was washed with H2O (3 x 50 mL) until complete removal of piperidine. The organic phase was dried over MgSO 4, filtered and the excess solvent was removed by rotary evaporation under reduced pressure to provide the crude product 27 which was used as such in the next step. LCMS 2.20 min (ES +) m / z (relative intensity) 964 ([M + H] +, 5%). (e) 6- (2,5-dioxo-2, 5-dihydro-lH-pyrrol-1-yl) -N- ((S) -l- (((S) -l - ((4- (( 3) -7-methoxy-8- (3- (((S) -7-methoxy-2- (4- (4-methylpiperazin-1-yl) phenyl) -5-oxo-5, lla-dihydro-1H -pyrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -5-oxo-5, lla-dihydro-lH-pyrrolo [2, 1-c] [1, 4] benzodiazepin-2-yl) phenyl) amino) -l-oxopropan-2-yl) amino) -3-methyl-l-oxobutan-2-yl) hexanamide (28) EDCI hydrochloride (4.7 mg, 0.03 mmol) was added to a suspension of 6-maleimidohexanoic acid (6.5 mg, 0.03 mmol) in dry CH2Cl2 (3 mL) under an argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 27 (34 mg, crude) was added. Stirring was maintained until the reaction was complete (6 hours). The reaction was diluted with CH2C12 and the organic phase was washed with H2O and saline before being dried over MgSO4, filtered and the excess solvent was removed by rotary evaporation under reduced pressure. The product was carefully purified by silica gel chromatography (slow elution starting with 100% CHCI3 to 9: 1 CHCls / MeOH) followed by reverse phase chromatography to remove maleimide-PEG8-unreacted acid. The product 28 was isolated in 41% on two steps (14.6 mg). LCMS 2.40 min (ES +) m / z (relative intensity) 1157 ([M + H] +, 5%).
Example 4 - Alternative synthesis of compound 25 PBD-triflate 21 (469 g, 0.323 mmol), boronic pinacol ester (146.5 mg, 0.484 mmol) and Na2CO3 (157 mg, 1. 48 mmol) were dissolved in a toluene / MeOH / H2O mixture, 2: 1: 1 (10 mL). The reaction flask was purged with argon three times before tetrakis (triphenylphosphine) palladium (0) (7.41 mg, 0.0064 mmol) was added and the reaction mixture was heated at 30 ° C overnight. The solvents were removed under reduced pressure and the residue was taken up in H20 (50 i) and extracted with EtOAc (3 x 50 mL). The combined organics were washed with saline (100 ml), dried with MgSO 4, filtered and the volatiles were removed by rotary evaporation under reduced pressure. The crude product was purified by gel column chromatography of silica (100% CHCl 3 at 95%: 5% CHCl 3 / MeOH) to provide pure 25 in a 33% yield (885 mg).
LC / MS 3.27 min (ES +) m / z (relative intensity) 1478 ([M + H] \ 100%).
Example 5 ' . (a) (S) -2- (4-Aminophenyl) -8- (3- (((S) -2- (benzo [d] [1,3] dioxol-5-yl) -7-methoxy -5 , 11-dioxo-10- ((2- (trimethylsilyl) ethoxy) methyl) -5, 10, 11, lla-tetrahydro-lH-plrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -7-methoxy-10- ((2- (trimethylsilyl) ethoxy) methyl) -IH-pyrrolo [2, 1-c] [1,4] benzodiazepine-5, 11 (10H, llaH) -dione (29) 3, 4- (Methylenedioxy) phenyl boronic acid (356 mg, 2.1 mmol, 1.3 equiv.), TEA (1.8 mL, 12.9 mmol, 8 equiv.) And triflate / aniline 13 (1.75 g, 1.7 mmol, 1 equiv.) were dissolved in a mixture of ethanol (7 ml), toluene (13 ml) and water (2 ml) under an atmosphere of Ar. The reaction mixture was evacuated and purged with Ar 3 times, before the addition of tetrakis (triphenylphosphine) palladium (0) (114 mg, 0. 1 mmol, 0.06 equiv.). The flask was again evacuated and purged with Ar 3 times and heated in a microwave at 80 ° C for 8 minutes with 30 seconds of pre-agitation time. Analysis by TLC (80:20 v / v ethyl acetate / hexane) indicated the complete consumption of the starting material. The reaction mixture was diluted with dichloromethane (50 ml) and washed with water (50 ml). The organic layer was dried with MgSO 4, filtered and the solvent was removed in vacuo. Purification by silica gel column chromatography (60:40 to 20:80 v / v hexane / ethyl acetate) gave product 29 as a yellow solid (1.21 g, 71%). LC / MS (3.92 min (ES +) m / z (relative intensity) 1032.44 ([M + H] +, 100). (b) (S) -2- (4-Aminophenyl) -8- (3- (((S) -2- (benzo [d] [1,3] dioxol-5-yl) -7-methoxy-5 -oxo-5, lla-dihydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -7-methoxy-lH-pyrrolo [2, 1 -c] [ 1,4] benzodiazepine-5 (llaH) -one (30) Dilactam SEM 29 (0.25 g, 0.24 mmol, 1 equiv.) Was dissolved in THF (8 mL) and cooled to -78 ° C under an atmosphere of Ar. Super-Hydride® (0.6 ml, 1M in THF, 2.5 equiv.) Was added dropwise during 5 minutes while temperature was monitored. After 20 minutes, a small sample was taken and prepared for the LCMS analysis. Water (50 ml) was added, the cold bath was removed and the solution was washed with ethyl acetate (50 i). The organic layer was extracted and washed with saline (60 ml), dried with MgSO 4, filtered and the solvent was removed in vacuo. The crude product was dissolved in EtOH (15 mL), CH2Cl2 (7.5 mL) and water (2.5 mL) and sufficient silica gel was added until it was a slurry. After 5 days of stirring, it was filtered through a sintered funnel and washed with 0H2012 / MQ0H (9: 1) (100 ml) until the product was no longer eluted. The organic layer was washed with saline (2 x 50 i), dried with MgSO 4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (CHCl 3 with a gradient of MeOH from 1% to 4%) provided the product 30 as a yellow solid (94 mg, 53%). LC / MS (2.53 min (ES +) m / z (relative intensity) 739.64 ([M] 70). (c) ((S) -l- (((S) -1 - ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1,3]] dioxol-5-yl) -7-methoxy-5-oxo-5, lla-dihydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -7-methoxy -5-oxo-5, lla-dihydro-lH-pyrrolo [2, 1-c] [7,4] benzodiazepin-2-yl) phenyl) amino) -1-oxopropan-2-yl) amino) -3- allyl methyl-l-oxobutan-2-yl) carbamate (3D Under an atmosphere of Ar, Alanine-Valine-Alloc (180 mg, 0.66 mmol, 1.2 equiv.) Was stirred with EEDQ (163 mg, 0.66 mmol, 1.2 equiv.) In anhydrous CH 2 Cl 2 (21 mL) and methanol (1 i) for 1 hour. PBD 30 (407 mg, 0.55 mmol, 1 equiv.) Was dissolved in anhydrous CH 2 Cl 2 (21 mL) and methanol (1 mL) and added to the reaction. LC / MS after 5 days of agitation at room temperature showed the highest product formation. The solvent was removed in vacuo before purification by column chromatography (CH2C12 with gradient of MeOH from 1% to 6%) to yield product 31 as a yellow solid (184 mg, 34%). LCMS (2.95 min (ES +) m / z (relative intensity) 994.95 ([M + H] +, 60). (d) (S) -2-Amino-N- ((S) -1- ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1, 3] dioxol-5-yl) -7-methoxy-5-oxo-5, lla-dihydro-1 H -pyrrolo [2, 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) - 7-methoxy-5-oxo-5, lla-dihydro-lH-pyrrolo [2, 1- c] [1,4] benzodiazepin-2-yl) phenyl) amino) -1-oxopropan-2-yl) -3-me t i lbutylnamide (32) Imine 31 (100 mg, 0.1 mmol, 1 equiv.) Was dissolved in anhydrous DCM (10 mL) (with the aid of a drop of methanol to aid dissolution) under an atmosphere of Ar. Pyrrolidine (30 ml, 0.15 mmol, 1.5 equiv.) Was added dropwise before the flask was evacuated and purged with Ar three times. Pd (PPh3) 4 (7 mg, 6 pmol, 0.06 equiv.) Was added and the flask was evacuated and purged with Ar three times. LC / MS analysis after 1 hour indicated product formation and complete loss of starting material. Et20 (60 ml.) Was added to the reaction mixture and allowed to stir until all the product had crashed out of the solution. The precipitate was filtered through a sintered funnel and washed twice with Et20 (2 x 20 mL). The collecting flask was replaced and the isolated solid was dissolved and washed through sintering with CHCl3 (100 ml). The solvent was removed in vacuo to give the crude product 32 as a yellow solid which was used directly in the next step. LC / MS (1.14 in (ES +) m / z (relative intensity) 910.40 ([M + H] +, 67). (e) N- ((S) -1- (((S) -l - ((4- ((S) -8- (3- (((S) -2- (Benzo [d] [1, 3] dioxol -5-yl) -7-methoxy-5-oxo-5,1-dihydro-lH-pyrrolo [2, 1 -c] [1,4] benzodiazepin 8 il) oxy) propoxy) -7- methoxy-5-oxo-5, l -díhidro-lH-pirrolo [2, 1 -c] [1,] benzodiazepin-2-yl) phenyl) amino) -1 -oxopropan-2-yl) amino) -3 -methyl-l -oxobutan-2-yl) -1 - (3- (2, 5-dioxo-2, 5-dihydro-lH-pyrrol-1-yl) propanamido) -3, b, 9, 12, 15 , 18, 21, 24-octaoxaheptacosan-27 -amide (33) Imine 32 (92 mg, 0.1 mmol, 1.1 equiv.) Was dissolved in CHCl3 (6 mL) with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEG8-acid (53 mg, 0.09 mmol, 1 equiv.) Was added followed by EEDQ (33 mg, 0.14 mmol, 1.5 equiv.). This was allowed to stir vigorously at room temperature under Ar for 4 days until the LC / MS analysis showed the largest product formation. The solvent was removed in vacuo and the crude product was partially purified by silica gel column chromatography (CHCl3 with gradient of MeOH from 1% to 10%) yielding 33 (81 mg). The material was further purified by preparative HPLC to give 33 as a yellow solid (26.3 mg, 18%). Rapid Formic Run: LC / MS (1.39 min (ES +) m / z (relative intensity) 1485. 00 ([M + H] +, 64).
Example 6 . .. (a) ((S) -l - (((S) -1- ((4- ((S) - 8 - (3- (((S) -2- (benzo [d] [1,3] dioxol -5-yl) -7-methoxy-5, 11-dioxo-l 0 - ((2- (trimethylsilyl) ethoxy) methyl) -5,10,11, lla-tetrahydro-lH-pyrrolo [2, 1- c] [1,] benzodiazepin-8-yl) oxy) propoxy) -7-methoxy-5,11-dioxo-l 0- ((2- (trimethylsilyl) ethoxy) methyl) -5, 10, 11, tetrahydro-lH-pyrolo [2, 1-c] [1,4] benzodiazepin-2-yl) f in i D amino) -1-oxopropan-2-yl) amino) -3-methyl-l -oxobutan- 2-yl) carbamate of 9H-fluoren-9-yl) methyl (34) Triflate 21 (0.5 g, 0.35 mmol, 1 equiv.), 3,4- (methylenedioxy) phenyl boronic acid (75 g, 0.45 mmol, 1. 3 equiv.) And Na 2 CO 3 (0.17 g, 1.6 mmol, 4.5 equiv.) Were dissolved in toluene (11 mL), EtOH (5.5 mL) and water (5.5 mL) under an atmosphere of Ar. The flask was evacuated and purged with Ar three times. Pd (PPh3) 4 (24 mg, 0.02 mmol, 0.06 equiv.) Was added and again the flask was evacuated and purged with Ar three times. This was heated to 30 ° C and left stirring overnight. The LC / MS analysis showed the complete loss of starting material. The solvent was removed in vacuo and the residue was dissolved in water (60 ml) before being washed with ethyl acetate (60 ml x 3). The combined organic layers were washed with saline (50 ml), dried with MgSO 4, filtered and the solvent removed in vacuo. Purification by column chromatography (50:50 to 25:75 v / v hexane / ethyl acetate) gave product 34 as a yellow solid (310 mg, 64%). LC / MS (1.44 min (ES) m / z (relative intensity) 1423.35 ([M-H], 79). (b) ((S) -l- (((S) -1 - ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1,3]] dioxol-5-yl) -7-methoxy-5-oxo-5, lla-dihydro-lH-p ir rolo [2, 1 -c] [1,] benzodiazepin-8-yl) oxy) propoxy) -7- methoxy-5-oxo-5, l-dihydro-lH-pyrrolo [2, 1-c] [1,4] benzodiazepin-2-yl) phenyl) amino) -1-oxopropan-2-yl) amino) - 3-methyl-1-oxobutan-2-yl) carbamic acid (9H-fluoren-9-yl) methyl ester (35) Dilactam SEM 34 (0.31 g, 0.22 mmol, 1 equiv.) Is dissolved in THF (10 mL) and cooled to -78 ° C under an atmosphere of Ar. Super-Hydride® (0.5 ml, 1M in THF, 2.5 equiv.) Was added dropwise during 5 minutes while temperature was monitored. After 30 minutes, a small sample was taken and prepared for the LC / MS analysis. Water (50 ml) was added, the cold bath was removed and the solution was washed with ethyl acetate (50 i). The organic layer was extracted and washed with saline (60 ml), dried with MgSO 4, filtered and the solvent was removed in vacuo. The crude product was dissolved in EtOH (13.2 ml), CH2Cl2 (6.6 ml) and water (2.2 ml) and sufficient silica gel was added until it was a slurry. After 5 days of stirring, it was filtered through a sinter funnel and washed with CH2Cl2 / MeOH (9: 1) (100 ml) until the product was no longer eluted. The organic layer was washed with saline (2 x 50 i), dried with MgSO 4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (CHCl3 with a gradient of MeOH from 1% to 4%) gave the pure product as a yellow solid (185 mg, 75%). LCMS (1.70 min (ES +) m / z (relative intensity) 1132.85 ([M + H] +, 60). (c) (S) -2-Amino-N- ((S) -1 - ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1, 3] dioxol-5-yl) -7-methoxy-5-oxo-5, l -dihydro-lH-pyrrolo [2, 1 -c] [1,] enzodiazepin-8-yl) oxy) propoxy) -7 - methoxy-5-oxo-5, lla-dihydro-lH-pyrrolo [2, 1-c] [1,] benzodiazepin-2-yl) phenyl) amino) -l-oxopropan-2-yl) -3-methylbutanamide ( 32) Ina 35 (82 mg, 0.07 mmol, 1 equiv.) Was dissolved in DMF (1 mL) before piperidine was added slowly (0.2 i, 2 mmol, excess). This solution was allowed to stir at room temperature for 20 minutes until the LC / MS analysis showed complete consumption of the starting material. The reaction mixture was diluted with CH2Cl2 (50 mL), washed with water (50 mL x 4), dried with MgSO4, filtered and the solvent removed in vacuo. The product 33 was used without further purification in the next step. LCMS (1.15 min (ES +) m / z (relative intensity) 910.60 ([M + H] +, 58).
General Experimental Methods for the Example 7 The progress of the reaction was monitored by thin layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminum plates. Visualization of TLC was achieved with UV light or iodine vapor unless otherwise indicated. Flash chromatography was performed using silica gel Merck Kieselgel 60 F254. Extraction and chromatography solvents were purchased and used without further purification by Fisher Scientific, UK.
All chemical products were purchased from Aldrich, Lancaster or BDH.
The spectra of 1R and 13C NMR were obtained in a Bruker Avance 400 spectrometer. Coupling constants are given in hertz (Hz). Chemical shifts are recorded in parts per million (ppm) downfield from tetramethylsilane. The spin multiplicities are described as s (singlet), bs (wide singlet), d (doublet), t (triplet), q (quartet), p (pentuplet) and m (multiplet). The IR spectra were recorded on a Perkin-Elmer FT / IR Paragon 1000 spectrophotometer by applying the sample in a chloroform solution using the "golden gate" ATR system. Optical rotations were measured at room temperature using a Bellingham and Stanlcy ADP 220 polarimeter. Mass spectrometry was performed on a ThermoQuest Navigator from Thermo Electron, spectra with Electrospray (ES) were obtained at 20 to 30 V. Accurate measurements of mass were performed using Micromass Q-TOF global tandem. All samples were run under an electrospray ionization mode using 50% acetonitrile in water and 0.1% formic acid as solvent. The samples were run in W mode which gives a typical resolution of 19000 in FWHH. The instrument was calibrated with [Glu] -Fibrinopeptide B immediately before the measurement.
CLEM LC / MS (Shimazu CLEM-2020) using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid).
Gradient: initial composition of 5% of B maintained for 0.25 min, then increase of 5% of B to 100% of B during a period of 2 min. The composition was maintained for 0.50 min at 100% B, and then it was returned to 5% B in 0.05 minutes and remained there for 0.05 min. The total run time of the gradient is equal to 3 min. Flow rate of 0.8 l / min. Wavelength detection interval: 190 to 800 nm. Oven temperature: 50 ° C. Column: Waters Acquity UPLC BEH Shield RP18 1.7 mm 2.1x50mm.
Preparative HPLC The conditions for preparative HPLC were as follows: HPLC (Shimadzu UFLC) was run using a mobile phase of water (0.1% formic acid) A and acetonitrile (0.1% formic acid) B. Wavelength detection interval : 254 nm.
Column: Phenomenex Gemini 5m C18150x21-20mm.
Gradient: B t 0 13% t = 15.00 95% t = 17.00 95% t = 17.10 13% t = 20.00 13% The total gradient run time is 20 minutes; flow rate of 20.00 ml / min.
Example 7 (a) Trifluoromethanesulfonate of (S) -5- (((tere-butyldimethylsilyl) oxy) methyl) -1- (5-methoxy-2-nitro-4- ((triisopropylsilyl) oxy) benzoyl) -4,5- dihydro-lH-pyrrol-3-yl (37) 2,6-Anhydrous lutidine (16.06 ml, 0.137 mol) was injected in one portion into a vigorously stirred solution of ketone 36 (20 g, 0.034 mol) in dry CH2Cl2 (350 ml) at -45 ° C (dry ice / acetonitrile) under an argon atmosphere. Anhydrous triflic anhydride, taken from a freshly opened bottle (17.37 ml, 0.1 mol), was injected rapidly, while maintaining the temperature at -40 ° C or below. The reaction mixture was allowed to stir at -45 ° C for 1 hour at which time TLC (Hexane / EtOAc; 95/5) revealed the complete consumption of the starting material. The cold reaction mixture was immediately diluted with CH2Cl2 (400 mL) and, with vigorous stirring, washed with ice-cold water (1 x 200 mL), 5% citric acid solution cooled with ice (1 x 300 i). , Saturated NaHCC (300 ml), saline solution (200 ml). The organics were dried over MgSO 4, filtered and the solvent was evaporated under reduced pressure. The crude material was purified by chromatography on silica gel (Hexane / EtOAc; 100% at 90:10) to provide enol triflate 37 as a yellow foam (22.06 g, 89%). 1 H-NMR (400 MHz, CDCl3) d 7.72 (s, 1H), 7.26 (s, 1H), 6.75 (s, 1H), 60.6 (bm, 1H), 5.75 (d, J = 5.7 Hz, 0.5H), 4.78 (m, 1H) , 4.59 (d, J = 8.2 Hz, 0.5H), 3.92 (s, 3H), 3.18 (dd, J = 15.2, 3.2 Hz, 4H), 2.99 (dd, J = 15.7, 3.2 Hz, 4H), 1.36 - 1.22 (m, 3H), 1.11 (d, J = 7.3 Hz, 18H), 0.92 (s, 9H), 0.12 (s, 6H); ES + = 2.39 min, m / z 1447.05 [2M + Na] +. (b) (11S) -8- (3-Bromopropoxy) -11- ((tert-butyldimethylsilyl) oxy) -2-cyclopropyl-1-methoxy-5-oxo-11, 11a-dihydro-1H-benzo [e] pyrr [1,2-a] [1,4] diazepine-10 (5H) -t-butylcarboxylate (45) I I Four. Five (i) (S) - (2- (((tert-Butyldimethylsilyl) oxy) methyl) -4-cyclopropyl-2,3-dihydro-lH-pyrrol-1-yl) (5-methoxy-2-nitro-4) - ((triisopropylsilyl) oxy) phenyl) methanone (38) Trifenilarsine (0.343 g, 1.12 mmol), silver oxide (I) (1.3 g, 5.6 mol), cyclopropylboronic acid (0.6 g, 7.01 mol) and triflate 37 (1 g, 1.4 mmol) were dissolved in dioxane (20 ml) under an argon atmosphere. Triassic potassium phosphate (3.6 g, 16.8 min) was ground with a pestle and mortar and added quickly to the reaction mixture. The reaction mixture was evacuated and purged with argon three times and heated to 71 ° C. Bis (benzonitrile) palladium (II) chloride (107 mg, 0.28 mmol) was added and the reaction vessel was evacuated and purged with argon three times. After 10 minutes, a small sample was taken for analysis by TLC (Hexane / EtOAc, 80:20) which revealed that the reaction had been completed. The reaction mixture was filtered through celite and the filter pad was washed with EtOAc (200 mL). The filtrate was washed with water (200 ml) and saline (200 ml). The organic layer was dried with MgSCq, filtered and the solvent removed in vacuo. Purification by chromatography on silica gel (Hexane / EtOAc: 100% at 80:20) gave product 38 as a yellow solid (0.663 g, 78%). 1 H-RN (400 MHz, CDCl 3) d 7.70 ( s, 1H), 7.33 (s, 1H), 6.77 (s, 1H), 4.64 (m, 1H), 3.90 (s, 3H), 3.70 (s, 2H), 2.64 (dd, J = 16.2, 2.42 Hz, 1H), 2.42 (dd, J = 16.2, 2.4 Hz, 1H), 1.35 - 1.22 (m, 3H), 1.19 (m, 1H), 1.10 (d, J = 7.3 Hz, 18H), 0.91 (s, 9H), 0.61 (m, 2H), 0.40 (dd, J = 7.2, 3.4 Hz, 2H), 0.10 (d, J = 1.9 Hz, 6H); ES + = 2.39 min, m / z 605.30 [M + H] +. (ii) (S) - (2-amino-5-methoxy-4- ((triisopropylsilyl) oxy) phenyl) (2- (((tert-butyldimethylsilyl) oxy) methyl) -4-cyclopropyl-2,3-dihydro -lH-pyrrol-l-yl) methanone (39) In a dry-bottomed two-neck round bottom flask with argon and equipped with a thermometer, nitrophenyl 38 (3.03 g 5 mol) was solubilized in a 5% formic acid solution in methanol (25 ml). Zinc (1.64 g, 25 immoles) was quickly poured into the solution. The temperature was instantaneously raised to 45 ° C and cooled slowly to return to room temperature at which time the reaction was completed (¾15 min, the reaction was monitored by LCMS). The reaction mixture was then filtered through celite and the pad was further washed with EtOAc (2 x 100 mL). The combined organics were subsequently washed with NaHCO 3. { ac) saturated (100 ml), H2O (100 ml) and saline (100 ml), before being dried over MgSO4, filtered and the volatiles were removed in vacuo. The raw material was purified by chromatography on silica gel (Hexane / EtOAc; 100% at 80:20) and the pure product 39 was isolated as a clear, colorless oil (1.35 g, yield 47%). 1 H-NMR (400 MHz, CDCl 3) d 7.26 (s, 2 H), 6.71 (s, 1 H), 4.61 (bs, 1 H), 4. 22 (s, 2H), 3.88 (s, 1H), 3.77 (s, 1H), 3.71 (s, 3H), 2.60 (dd, J = 16.5, 3.7 Hz, 1H), 2.43 (dd, J = 16.5, 3.7 Hz, 1H), 1.35 (m, 1H), 1.22 (m, 3H), 1.09 (d, J = 7.2 Hz, 18H), 0.89 (s, 9H), 0.68 - 0.58 (m, 2H), 0.48 - 0.36 (, 2H), 0.05 (d, J = 5.8 Hz, 6H). ES + = 2.40 min, m / z 575.30 [M + H] +. (iii) (S) - (2- (2- (((tert-butyldimethylsilyl) oxy) methyl) -4-cyclopropyl-2,3-dihydro-lH-pyrrole-l-carbonyl) -4-methoxy-5- (tere-butyl ((triisopropylsilyl) oxy) phenyl) carbamate (40) Amine 39 (770 mg, 1.34 ol) and Boc20 (350 mg, 1.6 mmol) were heated together at 70 ° C in a round bottom flask. To assist with solubility, CHCl3 (3 mL) was added and the mixture was allowed to stir until the reaction was complete (followed by LCMS). The crude thick solution was allowed to cool to room temperature before being loaded directly onto a silica gel chromatography column (Hexane / EtOAc: 100% at 95: 5). The product 40 was isolated as a colorless foam (741 mg, 82% yield). 1 H-NMR (400 MHz, CDCl 3) d 7.73 (s, 1H), 7.26 (s, 2H), 6. 73 (s, 1H), 4.64 (s, 1H), 3.91 (s, 1H), 3.78 (s, 1H), 3. 74 (s, 3H), 2.61 (dd, J = 16.2, 3.0 Hz, 1H), 2.45 (dd, J = 16.2, 3.0 Hz, 1H), 1.47 (s, 9H), 1.36 (m, 1H), 1.33 -1.23 (m, 3H), 1.11 (d, J = 7.3 Hz, 18H), 0.89 (s, 9H), 0.64 (m, 2H), 0.43 (m, 2H), 0.05 (d, J = 7.2 Hz, 6H); ES + = 2.56 min, m / z 675.30 [M + H] +. (iv) (S) - (2- (4-cyclopropyl-2- (hydroxymethyl) -2,3-dihydro-lH-pyrrole-1-carbonyl) -4-methoxy-5- ((triisopropylsilyl) oxy) phenyl) tert-butyl carbamate (4D Silyl ether 40 (741 mg, 1.1 mmol) was solubilized in a 7: 2: 1: 1 mixture of AcOH / H2O / MeOH / THF (11 mL) and the mixture was stirred at room temperature until the reaction was complete ( "3 hours). The volatiles were removed in vacuo and the residue was taken up in EtOAc (50 ml). The organic phase was washed with saturated NaHC03 (aq) (50 ml), H2O (50 ml) and saline (50 ml) before being dried over MgSO4, filtered and concentrated in vacuo. The crude material was purified by chromatography on silica gel (Hex / EtOAc; 100% at 60:40) and the pure product 41 was isolated as a colorless foam (521 mg, 84% yield). 1 H-NMR (400 MHz , CDCl3) d 7.94 (s, 1H), 7.65 (s, 1H), 6.75 (s, 1H), 6.17 (s, 1H), 4.71 (s, 1H), 4.60 (s, 1H), 3.82 (t, J = 8.7 Hz, 1H), 3.76 (s, 3H), 3.72 (d, J = 8.7 Hz, 1H), 2.76 (ddd, J = 16.4, 10.2, 1.6 Hz, 1H), 2.08 (dd, J = 16.3, 4.4 Hz, 1H), 1.48 (s, 9H), 1.42 - 1.33 (m, 1H), 1.33 - 1.23 (m, 3H), 1.11 (d, J = 7.3 Hz, 18H), 0.67 (dd, J = 5.0, 3.1 Hz, 2H), 0.46-0.39 (m, 2H); ES + = 2.25 min, m / z 561.45 [M + H] +. (v) (US) -2-cyclopropyl-11-hydroxy-7-methoxy-5-oxo-8- ((triisopropylsilyl) oxy) -11, lla-dihydro-lH-benzo [e jpirrolo [1, 2-a ] [1, 4] diazepine-10 (5H) -tert-butylcarboxylate (42) DMSO (163 mL, 2.29 mmol) was added to a cooled solution of oxalyl chloride (93 mL, 1.1 mmol) in CH 2 Cl 2 (2 mL) at -78 ° C. After 15 minutes, a solution of alcohol 41 (515 mg, 0.91 mmol) in CH2Cl2 (5 mL) was added dropwise to the oxidant mixture. The reaction was allowed to stir at -78 ° C for 1 hour before Net3 (640 μm, 4.59 mmol) was added and the mixture was allowed to warm to room temperature. After completion, the reaction mixture was diluted with CH2C12 (40 mL) and the solution was washed with 0.1 M HCl (aC) (50 mL), H2O (50 mL), NaHCC > 3 (aq) saturated (50 ml) and saline (50 ml). The organics were dried with MgSCg, filtered and the volatiles were removed in vacuo. The crude material was purified by chromatography on silica gel (Hexane / EtOAc: 100% at 60:40) to provide pure 42 as a white foam (350 mg, 68%). 1 H-NMR (400 MHz, CDCl 3) d 7.17 (s, 1H), 6.74 (s, 1H), 6.67 (s, 1H), 5.65 (dd, J = 8.6, 2.3 Hz, 1H), 3.85 (s, 3H), 3.77 (dt, J = 13. 2, 6.7 Hz, 1H), 3.38 (s, 1H), 2.88 (dd, J = 17.7, 9.2 Hz, 1H), 2.52 (d, J = 14.5 Hz, 1H), 1.46 (m, 1H), 1.39 (s, 9H), 1.31-1.19 (m, 3H), 1.10 (dd, J = 7.4, 2.1 Hz, 18H), 0.77-0.70 (m, 2H), 0.56-0.48 (m, 2H); ES + = 1.89 min, m / z 559.45 [M + H] +. (vi) (11S) -11- ((tert-Butyldimethylsilyl) oxy) -2-cyclopropyl-7-methoxy-5-oxo-8- ((triisopropylsilyl) oxy) -11, lla-dihydro-lH-benzo [ejirirole] [1, 2-a] [1,4] diazepine-10 ( 5H) -tert-butyl carboxylate (43) Alcohol 42 (350 mg, 0.62 mmol) was solubilized in dry CH 2 Cl 2 (5 mL) in a sealed round bottom flask pre-purged three times with argon. The solution was cooled to 0 ° C before the addition of lutidine (0.3 ml, 2.5 mmol) and TBS-OTf (0.43 ml, 1.8 mmol). The reaction mixture was allowed to warm to room temperature and stirred until complete (monitored by LCMS). After completion, the solution was diluted with CH2Cl2 (50 mL), washed with saturated NH4Cl (aq) (50 i), H2O (50 mL), NaHCC > 3 (aq) saturated (50 ml) and saline (50 ml). The organics were dried with MgSO 4, filtered and the volatiles were removed in vacuo. The crude material was purified by chromatography on silica gel (Hexane / EtOAc, 100% at 80:20) to give pure 43 as a colorless oil (397.3 mg, 94%). 1 H-RN (400 MHz, CDCl 3) d 7.16 (s, 1H), 6.69 (s, 1H), 6.63 (s, 1H), 5.78 (d, J = 9. 0 Hz, 1H), 3.84 (s, 3H), 3.65 (td, J = 10.1, 3.7 Hz, 1H), 2.82 (ddd, J = 16.8, 10.3, 2.6 Hz, 1H), 2.30 (dd, J = 16. 8, 2.6 Hz, 1H), 1.45 - 1.37 (m, 1H), 1.32 (s, 9H), 1.25 (dd, J = 14.1, 8.0 Hz, 3H), 1.09 (dd, J = 7.4, 4.1 Hz, 18H), 0.85 (s, 9H), 0.74 - 0.67 (m, 2H), 0.55 - 0.49 (m, 1H), 0.47-0.40 (m, 1H), 0.25 (s, 3H), 0.20 (s, 3H); ES + = 2. 39 min, m / z 695.55 [M + Na] +. vii) (US) -11- ((tert-butyldimethylsilyl) oxy) -2-cyclopropyl-8-hydroxy-7-methoxy-5-oxo-ll, lla-dihydro-lH-benzo [e jpirrolo [1, 2- a] [1,4] diazepine-10 (5H) -t-butylcarboxylate (44) Monomer 43 (518.8 mg, 0.77 mmol) was solubilized in humid DMF (5 ml + 0.1 ml H2O) before LiOAc (78.5 mg, 0.77 mmol) was added and the mixture was allowed to stir at room temperature until complete (continued) of CLEM). The mixture was subsequently diluted with EtOAc (50 mL), quenched with citric acid (aq.) (PH = 3.40 mL), then washed with H20 (50 mL) and saline (50 mL). The organic layer was dried over MgSO 4, filtered and the volatiles were removed in vacuo. The crude product was purified by chromatography on silica gel (Hexane / EtOAc; 100% at 60:40) and the pure product 44 was isolated as a white solid (351 mg, 88% yield). 1 H-NMR (400 MHz, CDCl 3) d 7.20 (s, 1 H), 6.68 (s, 1 H), 6.68 (s, 1 H '), 5.79 (d, J = 8.9 Hz, 1 H), 3.94 (s, 3 H) , 3.70 (td, J = 10.1, 3.7 Hz, 1H), 2.82 (ddd, J = 16.9, 10.3, 2.0 Hz, 1H), 2.31 (dd, J = 16.9, 2.0 Hz, 1H), 1.44 1.37 (m, 1H), 1.32 (s, 9H), 0.86 (s, 9H), 0.75 - 0.68 (m, 1H), 0.57 - 0.49 (m, 1H), 0.46 (m, 1H), 0.23 (d, J = 6.9 Hz, 6H); ES + = 1.82 min, m / z 517.35 [M + Na] +. (viii) (11S) -8- (3-Bromopropoxy) -11 - ((tert-butyldimethylsilyl) oxy) -2-cyclopropyl-7-methoxy-5-oxo-ll, 11a-dihydco-lH-benzo [e] pyrrolo [1, 2-a] [1,4] diazepine-10 (5H) -t-butylcarboxylate (45) In a dry round-bottomed flask pre-purged three times with argon, alcohol 44 (300 mg, 0. 58 mmol) was solubilized in dry DMF (5 ml). K2CO3 (123 mg, 0.58 mmol) and 1,3-dibromopropane (0.3 ml, 2.9 mmol) were added later. The reaction mixture was heated to 70 ° C and left to stir until complete (1 hour, followed by LCMS). The reaction was diluted with EtOAc (50 mL), washed with H2O (75 mL) and saline (50 mL) before being dried over MgSO4, filtered and the volatiles removed in vacuo. The crude material was purified by chromatography on silica gel (Hexane / EtOAc: 100% at 70:30) and the pure product 45 was isolated as a colorless foam.(311 mg, 84% yield) 1 H-NMR (400 MHz, CDCl 3) d 7. 21 (s, 1H), 6.69 (s, 1H), 6.63 (s, 1H), 5.82 (d, J = 8.8 Hz, 1H), 4.14 (t, J = 5.9 Hz, 2H), 3.90 (s, 3H), 3.69 (ddd, J = 10.2, 9.0, 3.7 Hz, 1H), 3.63 (t, J = 6.3 Hz, 2H), 2.84 (ddd, J = 16.7, 10.4, 1.9 Hz, 1H), 2.38 (p, J = 6.1 Hz, 2H), 2.31 (dd, J = 16.5, 2.1 Hz, 1H), 1.45 - 1.37 (m, 1H) 1.33 (s, 9H), 0.87 (s, 9H), 0.77 - 0.69 (m, 2H), 0.57 0.49 (m, 1H), 0.49-0.42 (m, 1H), 0.24 (d, J = 5.4 Hz 6H); ES + = 2.16 min, m / z 638.95 [M + Na] +. (c) (11S) -2- (4- ((S) -2 - ((S) -2 - ((((9H-Fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamide) phenyl) -11- ((tert-butyldimethylsilyl) oxy) -8-hydroxy-7-methoxy-5-oxo-ll, 11a-dihydro-lH-benzo [e Jpirrolo [1, 2-a] [1, 4] Diazepine-10 (5H) -tert-butylcarboxylate (53) - l - - - l - - Í; - l - - l - - (i) (S) - (4 - (4-aminophenyl) -2- (((terebutyldimethylsilyl) oxy) methyl) -2,3-dihydro-lH-pyrrol-1-yl) (5-methoxy-2- ni tro-4- ((triisopropylsilyl) oxy) phenyl) methanone (46) Pd (PPh3) 4 (609 mg, 0.52 mmol) was added to a stirred mixture of triflate 37 (18.8 g, 26.3 moles), 4-aminophenylboronic acid pinacol ester (8.64 g, 39.4 mmol), Na2CO3 (12.78 g, 120 mmol), MeOH (80 ml), toluene (160 ml) and water (80 ml). The reaction mixture was allowed to stir at 30 ° C under a nitrogen atmosphere for 24 hours after which all the boronic ester was consumed. The reaction mixture was then evaporated to a point before the residue was taken up in EtOAc (100 mL) and washed with H2O (100 mL), saline (100 mL), dried (MgSO4), filtered and evaporated under reduced pressure to provide the crude product. Purification by chromatography on silica gel (Hexane / EtOAc, 100% at 70:30) gave the product 46 as a yellowish foam (11.06 g, 64%). 1 H-NMR (400 MHz, CDCl 3) d 7.74 (s, 1H), 7.00 (d, J = 8.3 Hz, 2H), 6.81 (s, 1H), 6.58 (d, J = 8.3 Hz, 2H), 6.06 (s, 1H), 4.77 (bm, 1H), 3.91 ( d, J = 6.7 Hz, 3H), 3.68 (bs, 2H), 3.13 (bm, 1H), 2.97 (d, J = 14.5 Hz, 1H), 1.36-1.21 (m, 3H), 1 .12 (d, J = 7.3 Hz, 18H), 0.89 (s, 10H), 0.10 (s, 6H); ES + = 2 . 27 ruin, m / z 698 [M + CH3CN] +. (ü) ((S) -l - (((S) -l - ((4- ((S) -5- (((tert-butyldimethylsilyl) oxy) methyl -1 - (5-methoxy-2-ni tro-4- ((triisopropylsilyl) oxy) benzoyl) -, 5-dihydro-lH-pyrrol-3-yl) phenyl) amino) -l -oxopropan-2-yl) amino) -3-methyl-l -oxobutan- 2-yl) carbamate of (9H-fluoren 9-yl) methyl (47) To a dry round bottom flask purged previously with argon was added aniline 46 (10.05 g, 15.3 mmol), the dipeptide (6.3 g, 15.3 mmol) and dry CH2Cl2 (500 mL). The flask was then purged three times with argon before EEDQ (3.79 g, 15.3 mmol) was added and the mixture was allowed to stir at room temperature. The reaction was followed by LCMS and after 3.5 hours the reaction was complete. The reaction was quenched with H2O (200 mL) and extracted twice with CH2C12 (250 mL). The combined organics were washed with saline (150 ml), dried over MgSO 4, filtered and the solvent removed in vacuo. The crude product was purified by chromatography on silica gel (Hexane / EtOAc; 100% at 55:45) to give the pure product 47 (13.821 g, 86%). X H - NMR (400 MHz, CDCl 3) d 8.26 (s, 1 H), 7.64 (s + d, J = 4.9 Hz, 3H), 7.43 (t, J = 7.3 Hz, 1H), 7.36 (d, J = 7.3 Hz, 1H), 7.28 (t, J = 7.3 Hz, 1H), 7.19 (d, J = 7.7 Hz, 1H), 6. 99 (d, J = 7.9 Hz, 1H), 6.71 (s, 1H), 6.27 (d, J = 6.3 Hz, 1H), 6.08 (s, 1H), 5.11 (d, J = 6.6 Hz, 1H), 4.69 (bs, 1H), 4.52 (bm, 1H), 4.36 (d, J = 6.5 Hz, 2H), 4.08 (t, J = 5. 9 Hz, 1H), 3.89 (m, 1H), 3.80 (s, 3H), 3.11 - 2.97 (bm, 1H), 2.88 (bd, J = 15.2 Hz, 1H), 2.03 (bs, 1H), 1.33 (d, J = 6.9 Hz, 3H), 1.24 - 1.11 (m, 3H), 1.01 (d, J = 7.4 Hz, 18H), 0.86-0.79 (m, 6H), 0.77 (s, 9H), 0.00 (s, 6H); ES + = 2.37 min, without mass. (iii) ((S) -1- (((S) -l- ((4- ((S) -1- (2-amino-5-methoxy-4- ((triisopropylsilyl) oxy) benzoyl) -5 - (((tert-butyldimethylsilyl) oxy) methyl) -4,5-dihydro-lH-pi rro 1-3-yl) phenyl) amino) -l-oxopropan-2-yl) amino) -3-methyl-1 [9H-fluoren-9-yl] methyl (oxobutan-2-yl) carbamate (48) In a dry two-neck round bottom flask pre-purged with argon and equipped with a thermometer, nitrophenyl 47 (2.97 g, 2.8 moles) was solubilized in a 5% formic acid solution in methanol (50 ml). Zinc (1.85 g, 28 mmol) was quickly poured into the solution. The temperature was instantaneously raised to 40 ° C and cooled slowly to return to room temperature at which time the reaction was complete ("15 minutes, the reaction was monitored by LCMS). The reaction mixture was filtered through celite and the pad was further washed with EtOAc (2 x 150 mL). The combined organics were subsequently washed with saturated NaHC03 (aq) (100 ml), H20 (100 ml) and saline (100 ml), before dried over MgSO4, filtered and the volatiles were removed in vacuo. The crude material was purified by chromatography on silica gel (Hexane / EtOAc: 75:25 to 50:50) and the pure product 48 was isolated as a light yellow oil (2291 g, 79% yield). NMR (400 MHz, CDCl3) d 8.37 (s, 1H), 7.74 (s + d, J = 4.9 Hz, 3H), 7.53 (t, J = 7.4 Hz, 2H), 7.46 (d, J = 11.3 Hz, 2H), 7. 39 (t, J = 7.3 Hz, 2H), 7.28 (t, J = 11.3 Hz, 2H), 7.09 (d, J = 7.9 Hz, 2H), 6.38 (d, J = 6.3 Hz, 1H), 6.18 (s, 1H), 5.21 (d, J = 2.9 Hz, 1H), 4.81 (bs, 1H), 4.72 - 4.57 (m, 1H), 4.47 (d, J = 6.5 Hz, 2H), 4.19 (t, J = 5.0 Hz, 1H), 4.00 - 3.94 (m, 1H), 3.91 (s, 3H), 3.23 - 3.07 (m, 1H), 2.98 (d, J = 16.8 Hz, 1H), 2.15 (s, 1H), 1.43 (d, J = 6. 9 Hz, 3H), 1.36 - 1.18 (m, 3H), 1.12 (d, J = 7.4 Hz, 18H), 0.97-0.89 (m, 6H), 0.88 (s, 9H), 0.10 (s, 6H). ES + = 2.37 min, m / z without mass. (iv) ((S) -l - (((S) -1 - ((4- ((S) -l- (2- ((tere-butoxycarbonyl) amino) -5-methoxy-4 - ((triisopropylsilyl ) oxy) benzoyl) -5- (((tert-butyldimethylsilyl) oxy) methyl) -4,5-dihydro-lH-pyrrol-3-yl) phenyl) amino) -l -oxopropan-2-yl) amino) - 3-methyl-l-oxobutan-2-yl) carbamate (9H-fluoren-9-yl) methyl (49) Amine 48 (14,913 g, 14.6 mmol) and Boc 20 (3.83 g, 17.5 mmol) were heated together at 70 ° C in a round bottom flask. To help with the solubility, it was added CHCl 3 (25 mL) and the mixture was allowed to stir until the reaction was complete (followed by LCMS). The crude thick solution was allowed to cool to room temperature before being loaded directly onto a silica gel chromatography column (Hexane / EtOAc, 100% at 65:35). The product 49 was isolated as a cream foam (13.2 mg, 80% yield). 1 H-NMR (400 MHz, CDCl 3) d 8.40 (s, 1H), 8.21 (s, 1H), 7.74 (d, J = 7.8 Hz, 3H), 7.54 (t, J = 7. 0 Hz, 2H), 7.48 (d, J = 7.7 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.31 - 7.25 (m, 3H), 7.14 (d, J = 6.7 Hz, 2H), 6.84 (bs, 1H), 6.80 (s, 1H), 6.50 (d, J = 6.4 Hz, 1H), 5.28 (d, J = 6.0 Hz, 1H), 4.77 (d, J = 2.6 Hz, 1H), 4.70 - 4.58 (m, 1H), 4.47 (t, J = 5.7 Hz, 2H), 4.19 (t, J = 6.1 Hz, 1 HOUR), 4. 00 (m, 2H), 3.88 (bs, 1H), 3.73 (s, 3H), 3.05 (m, 1H), 2. 98 (dd, J = 15.4, 3.3 Hz, 1H), 2.15 (bm, 1H), 1.46 (s, 9H), 1.43 (d, J = ll.7 Hz, 3H), 1.36-1.22 (m, 3H), 1.12 (d, J = 7.4 Hz, 18H), 1.00-0.89 (m, 6H) , 0.84 (s, 9H), 0.05 (d, J = 6.0 Hz, 6H); ES + = 2.53 min, without mass. (v) ((S) -l (((S) 1 - ((4- ((S) -1 - (2 - ((tere-bu toxic rbonyl) amino) -5-methoxy -4- ((triisopropylsilyl ) oxy) benzoyl) -5- (hydroxymethyl) -4,5-dihydro-lH-pyrrol-3-yl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-l -oxobutan- 2-yl) (9H-fluoren-9-yl) methyl carbamate (50) Silyl ether 49 (13.2 g, 11.8 mmol) was solubilized in a 7: 2: 1: 1 mixture of AcOH / H2O / MeOH / THF (220 mL) and the mixture was stirred at room temperature until the reaction was complete (overnight). The volatiles were removed in vacuo and the residue was taken up in EtOAc (400 ml). The organic phase was washed with saturated NaHCO3 (aq) (200 i), H20 (200 ml) and saline (10 ml) before being dried over MgSO4, filtered and concentrated in vacuo. The crude material was purified by chromatography on silica gel (Hex / EtOAc; 50:50 to 0: 100) and the pure product 50 was isolated as a light yellow foam (11168 mg, 94% yield). NMR (400 MHz, CDCl 3) d 8.45 (s, 1 H), 7.93 (s, 1 H), 7.74 (d, J = 7.4 Hz, 2 H), 7.64 (s, 1 H), 7.52 (dd, J = 17.9, 8.9 Hz, 4H), 7.39 (t, J = 7.4 Hz, 2H), 7.33 - 7.26 (m, 3H), 7.13 (d, J = 7.4 Hz, 2H), 6.81 (s, 1H), 6.45 (s, 1H), 5.26 (s, 1H), 4.84 (s, 1H), 4.69 -4.58 (m, 1H), 4.47 (d, J = 6.2 Hz, 2H), 4.43 (s, 1H), 4.17 (d, J = 14.2 Hz, 1H), 3.99 (s, 1H), 3.89 (s, 2H), 3.74 (s, 3H), 3. 30 - 3.17 (m, 1H), 2.64 (d, J = 16.9 Hz, 1H), 2.23 - 2.09 (, 1H), 1.44 (s, 9H), 1.44 (d, J = 10.9 Hz, 2H), 1.29 (ddd, J = 14.3, 13.0, 7.4 Hz, 3H), 1.12 (d, J = 7.4 Hz, 18H), 0.92 (m, 6H); ES + = 2.23 min, without mass. (vi) (US) -2- (4- ((S) -2- ((S) -2- ((((9H-Fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamide phenyl) -11-hydroxy-7-methoxy-5-oxo-8- ((tpropyl ester) oxy) -11, -dihydro-lH- benzo [e] pyrr [1, 2-a] [1,4] diazepine-10 (5H)-tere-butylcarboxylate (5D) DMSO (1.55 1, 21.9 mmol) was added to a cooled solution of oxalyl chloride (0.89 mL, 10.5 mmol) in CH 2 Cl 2 (50 mL) at -78 ° C. After 15 minutes, a solution of alcohol 50 (8.8 mg, 8.76 mmol) in CH2Cl2 (100 ml) was added dropwise to the oxidant mixture. The reaction was allowed to stir at -78 ° C for 1 hour before Net3 (6.11 ml, 43.8 mmol) was added and the mixture was allowed to warm to room temperature. After completion, the reaction mixture was diluted with CH2Cl2 (100 mL) and the solution was washed with HCl (aC) 0.1 M (250 mL), H2O (250 mL), NaHCC > 3 (aq) saturated (250 ml) and saline (200 ml). The organics were dried with MgSO 4, filtered and the volatiles were removed in vacuo. The crude material was purified by chromatography on silica gel (CH2Cl2 / EtOAc; 100% at 50:50) to give pure 51 as a yellow oil (8.8 mg, 100%). XH-NMR (400 MHz, CDCI3) d 8.71 (s, 1H), 7.74 (t, J = 8.4 Hz, 3H), 7.52 (d, J = 7.4 Hz, 5H), 7.43 - 7.33 (m, 4H), 7.23 - 7.17 (m, 2H), 6.69 (s, 1H), 6.42 (d, J = 7.9 Hz, 1H), 5.78 (d, J = 7. 8 Hz, 1H), 5.62 (s, 1H), 5.23 (d, J = 7.7 Hz, 1H), 4.84 -4.69 (m, 1H), 4.65 (d, J = 22.5 Hz, 1H), 4.45 - 4.29 ( m, 2H), 3.91 (dd, J = 11.3, 8.1 Hz, 1H), 3.86 (s, 3H), 3.28 (q, J = 11.9 Hz, 1H), 2.98 (t, J = 12.6 Hz, 1H), 2.14 (dd, J = 12.9, 10.0 Hz, 1H), 1.52 - 1.42 (m, 3H), 1.38 (s, 9H), 1. 26 (m, 3H), 1.16 - 1.05 (m, 18H), 0.93 (d, J = 6.0 Hz, 6H); ES + = 2.19 min, without mass. (vii) (11S) -2- (4- ((S) -2- ((S) -2- ((((9H-Fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamide ) phenyl) -11 - ((tert-butyldimethylsilyl) oxy) -7-methoxy-5-oxo-8- ((triisopropylsilyl) oxy) -11, lla-dihydro-lH-benzo [e Jpirrolo [1,2-a ] [1,4] diazepine-10 (5H) -t-butylcarboxylate (52) The alcohol 51 (8.8 g, 8.78 m oles) was solubilized in dry CH2Cl2 (150 ml) in a sealed round bottom flask pre-purged three times with argon. The solution was cooled to 0 ° C before the addition of lutidine (4 ml, 35.1 mmol) and TBS-OTf (6 ml, 26.3 mmol). The reaction mixture was allowed to warm to room temperature and stirred until complete (monitored by LCMS). After completion, the solution was diluted with CH2Cl2 (100 i), washed with saturated NH4Cl (aq) (150 ml), H2O (100 ml), saturated NaHC03 (aq) (100 i) and saline (100 ml). my). The organics were dried with MgSCg, filtered and the volatiles were removed in vacuo. The crude material was purified by chromatography on silica gel (Hexane / EtOAc: 100% at 80:20) to provide pure 52 as a colorless oil (6.18 mg, 70%). 1 H-NMR (400 MHz, CDC13) d 8.40 (s, 1 H), 7.76 (d, J = 7. 5 Hz, 2H), 7.55 (dd, J = 13.0, 6.7 Hz, 4H), 7.40 (t, J = 7. 3 Hz, 4H), 7.33 - 7.27 (m, 3H), 7.21 (s, 1H), 6.67 (s, 1H), 6.49 (s, 1H), 5.87 (d, J = 8.8 Hz, 1H), 5.30 (d, J = 5. 7 Hz, 1H), 4.71 - 4.59 (m, 1H), 4.48 (d, J = 6.8 Hz, 2H), 4.20 (t, J = 6.7 Hz, 1H), 4.04 - 3.96 (m, 1H), 3.86 ( s, 3H), 3.84 - 3.77 (m, 1H), 3.25 (m, 1H), 2.79 (d, J = 1.5 Hz, 1H), 2.26 - 2.11 (m, 1H), 1.46 (d, J = 6.9 Hz, 3H), 1. 33 (s, 9H), 1.27 (dd, J = 17.1, 9.7 Hz, 3H), 1.11 (dd, J = 7.4, 4.0 Hz, 18H), 0.93 (s, 6H), 0.89 (s, 9H), 0.27 (s, 3H), 0.22 (s, 3H); ES + = 2.55 min, m / z 116.30 [M + H] +. (viii) (11S) -2- (4- ((S) -2- ((S) -2 - ((((9H-Fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamide ) phenyl) -11- ((tert-butyldimethylsilyl) oxy) -8-hydroxy-7-methoxy-5-oxo-ll, 11a-dihydro-lH-benzo [e jpirrolo [1,2-a] [1,4 ] diazepine-10 (5H) -tert-butylcarboxylate (53) The monomer 52 (1 g, 0.89 mmol) was solubilized in Wet DMF (5 ml +0.5 ml H20) before LiOAc (91 mg, 0.89 mmol) was added and the mixture was allowed to stir at room temperature until complete (~ 3h, followed by CLEM). The mixture was subsequently diluted with EtOAc (50 ml), quenched with citric acid (aq) (pH = 3.40 ml), then washed with H2O (50 ml) and saline (50 ml). The organic layer was dried over MgSO 4, filtered and the Volatile were removed in vacuo. The crude product was purified by chromatography on silica gel (Hexane / EtOAc / MeOH: 60: 40: 0 to 60:30:10) and the pure product 53 was isolated as a cream-colored solid (675 mg, yield 78%). %). '-H-NMR (400 MHz, CDCl3) d 8.36 (s, 1H), 7.76 (d, J = 7.6 Hz, 2H), 7.55 (dd, J = 16.0, 7.5 Hz, 4H), 7.40 (t, J = 7.4 Hz, 4H), 7.30 (ddd, J = 14.7, 7.4 , 1. 1 Hz, 3H), 7.24 (s, 1H), 6.72 (s, 1H), 6.38 (d, J = 5.3 Hz, 1H), 5.87 (s, 1H), 5.23 (d, J = 6.2 Hz, 1H), 4.69 -4.57 (m, 1H), 4.49 (d, J = 6.6 Hz, 2H), 4.20 (t, J = 5.3 Hz, 1H), 4.04 - 3.96 (m, 1H), 3.96 (s, 3H), 3.87 (dd, J = 10. 1, 3.5 Hz, 1H), 3.29 (dd, J = 18.0, 8.5 Hz, 1H), 2.80 (d, J = 19.4 Hz, 1H), 2.24 - 2.08 (m, 1H), 1.46 (d, J = 10. 5 Hz, 3H), 1.33 (s, 9H), 1.00 - 0.91 (m, 6H), 0.90 (s, 9H), 0.25 (d, J = 8.6 Hz, 6H); ES + = 2.08 min, m / z 960.35 [M + H] +. (d) N- ((2 S) -1- (((2 S) -1- ((4- (8- (3- ((2-cyclopropyl-7-methoxy-5-oxo-5, dihydro-l-f-benzo [e] pyrr lo [1,2-a] [1,4] diazepin-8-yl) oxy) propo i) -7-methoxy-5-oxo-5,11a-dihydro-lf- benzo [e] pyrrolo [1,2-a] [1,4] diazepin-2-yl) phenyl) amino) -1- -2-yl) amino) -3-methyl-1-oxobutan-2-yl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamide) - 3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (18) Boc OTBS Boc OTBS - - . (i) (11S) -2- (4- ((S) -2- ((S) -2-amino-3-methylbutanamido) propanamido) phenyl) -8- (3- (((11S) -10- (terc- butoxycarbonyl) -11 - ((tert-butyldimethylsilyl) oxy) -2-cyclopropi-1-7-methoxy-5-oxo-5, 10, 11, lla-tetrahydro-lH-benzo [e Jpirrolo [1,2-a] [1, 4] dlazepin-8-ll) oxy) propoxy) -11 - ((tert-butyldimethylsilyl) oxy) -7-methoxy-5-oxo-ll, 11a-dihydro-lH-benzo [e] pyrr [ 1, 2-a] [1,4] diazepine-10 (5H) -carboxylate of tert-butyl (54) In a dry round-bottomed flask pre-purged three times with argon, the monomer 45 (310 mg, 0. 48m ol), monomer 53 (513 mg, 0.53 mmol), K2CO3 (103 mg, 0.48 mmol) and TBDI (18 mg, 0.048 mmol) were solubilized in dry DMF (5 ml) and the mixture was heated to 60 °. C. The reaction was allowed to stir until complete (followed by LCMS), before being diluted with EtOAc (50 mL), washed with H2O (75 mL) and saline (50 mL). The organics were dried over MgSO4, filtered and the volatiles were removed in vacuo. The crude material was purified by chromatography on silica gel (CHCls / MeOH: 100% to 98: 2) and the pure product 54 was isolated as a white solid (280.3 mg, 46% yield). X H-NMR (400 MHz, CDCl 3) d 8.93 (s, 1 H), 7.85 (d, J = 7.6 Hz, 1 H), 7.52 (d, J = 8.6 Hz, 2H), 7.40 (s, 1H), 7.28 (d, J = 8.6 Hz, 2H), 7.19 (s, 2H), 6.69 (s, 1H), 6.63 (s, 1H), 6.61 (s, 1H), 5.90 (d, J = 9. 3 Hz, 1H), 5.81 (d, J = 5.5 Hz, 1H), 4.60 (p, J = 7.1 Hz, 1H), 4.20 (dd, J = 15.9, 11.1 Hz, 4H), 3.88 (s, 3H) , 3.87 (s, 3H), 3.84 (dd, J = 6.3, 4.5 Hz, 1H), 3.68 (td, J 10. 2, 3.7 Hz, 1H), 3.38 - 3.22 (m, 2H), 2.89 - 2.73 (m, 2H), 2.48-2.26 (m, 4H), 1.47 (d, J = 7.0 Hz, 3H), 1.42 (m, 1H), 1.30 (s, 18H), 1.02 (d, J = 7.0 Hz, 3H), 0.89 (s, 9H), 0.86 (s, 10H), 0.84 (s, 6H), 0.72 (dd, J = 8.1, 3.3 Hz, 2H), 0.57 - 0.50 (m, 1H), 0.45 (m, 1H), 0.28 - 0.20 (m, 12H); ES + = 2.16 min, m / z 1297.55 [M + Na] +. (ii) (11S) -8- (3- (((11S) -10- (tert-butoxycarbonyl) -11 - ((tert-butyldimethylsilyl) oxy) -2- (4- ((2S, 5S) -31 - [2,5-dioxo-2, 5-dihydro-lH-pyrrol-l -yl) -5-isopropyl-2-methyl-4,7,35-trioxo-10, 13, 16, 19, 22, 25 , 28, 31 -octaoxa-3, b, 34-triazaheptatriacontanamido) phenyl) -7-methoxy-5-oxo-5, 10, 11, lla-tetrahydro-lH-benzo [e Jpirrolo [1, 2-a] [ 1,4] diazepin-8-yl) oxy) propoxy) -11 - ((tert-butyldimethylsilyl) oxy) -2-cyclopropyl-7-methoxy-5-oxo-ll, 11a-dihydro-lH-benzo [ejpirrolo [ 1, 2-a] [1,4] diazepine-10 (5H) -carboxylate of tert-butyl (55) In a dry round-bottomed flask pre-purged three times with argon, the dimer 54 (270 mg, 0. 021 mmol) was solubilized in dry CH 2 Cl 2 (6 mL). EDCI hydrochloride (40 mg, 0.021 mmol) and maleimide-PEG8-OH (123 mg, 0.021 mmol) were subsequently added to the solution which was allowed to stir at room temperature until complete (¾ 1 hour, followed by LCMS). After completion, the reaction was diluted with CH2Cl2 (50 mL) and the organic phase was washed with H2O (50 mL) and saline (50 mL). mi) before being dried over MgSO 4, filtered and the volatiles were removed by rotary evaporation under reduced pressure. The crude material was purified by chromatography on silica gel (CHCl3 / 100% MeOH to 97: 3) and the pure product 55 was isolated as a light yellow foam (318.8 mg, 82% yield). 1 H-NMR (400 MHz, CDCl 3) d 8.64 (s, 1 H), 7.69 (d, J = 15.0 Hz, 2H), 7. 39 (s, 1H), 7.28 (d, J = 15.0 Hz, 2H), 7.26 (s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 7.03 (d, J = 4.5 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 6.69 (s, 2H), 6.65 (s, 1H), 6.63 (s, 1H), 6. 37 (t, J = 4.7 Hz, 1H), 5.89 (d, J = 6.8 Hz, 1H), 5.81 (d, J = 8.4 Hz, 1H), 4.67 (p, J = 7.2 Hz, 1H), 4.27 - 4.12 (m, 5H), 3.88 (s, 3H), 3.87 (s, 3H), 3.84 (t, J = 5.6 Hz, 1H), 3.73 - 3.56 (m, 46H), 3.53 (t, J = 5.0 Hz, 1H), 3.41 (dd, J = 10.3, 5.2 Hz, 1H), 3.35 - 3.22 (m, 1H), 2.90 -2.74 (m, 1H), 2.67 (ddd, J = 13.6, 9.2, 4.1 Hz, 1H), 2.52 (t, J = 7.2 Hz, 1H), 2.48 - 2.45 (, 1H), 2.45 - 2.37 (m, 1H), 2.37-2.22 (m, 1H), 2.02 (t, J = 9.0 Hz, 1H), 1.45 (d, J = 7.1 Hz, 3H), 1.42 - 1.37 (m, 1H), 1.30 (s, 9H), 0. 99 (s, 6H), 0.89 (s, 9H), 0.86 (s, 9H), 0.78 - 0.67 (m, 2H), 0.58-0.49 (m, 1H) 0.48-0.42 (m, 1H), 0.28 - 0.20 (m, 12H); ES + = 2.15 min, m / z 1891.60 [M + Na] +. (iii) N- ((2S) -l - (((2S) -1 - ((4- (8 - (3- ((2-cyclopropyl-7-methoxy-5 -oxo-5, -dihydro) -lH-benzo [e] pyrr [1, 2-a] [1,4] diazepin-8-yl) oxy) propoxy) - 7- methoxy-5-oxo-5, l-dihydro-lH-benzo [e] pyrr lo [1, 2-a] [1,4] diazepin-2-yl) phenyl) amino) - l -oxopropan-2- il) amino) -3-methyl-l-oxobutan-2-yl) -1 - (3- (2, 5-dioxo-2, 5-dihydro-lH-pyrrol-1-yl) propanamido) -3, b , 9, 12, 15, 18, 21, 24-octaoxaheptacosan-27 -amide (18) Dimer 55 (318 mg, 0.017 inmol) was solubilized in dry H 2 O (160 ml) and the suspension was cooled to 0 ° C before TEA (4 ml) was added and the mixture was allowed to stir until complete (20 mis) , followed by CLEM). After completion, the reaction was diluted with CH2Cl2 (50 mL) and the organic phase was washed with ice-cold NaHC03 (2 x 50 mL), H20 (50 mL) and saline (50 mL) before being dried over MgSO4, it was filtered and the volatiles were removed in vacuo. The crude material was purified directly by reverse phase preparative HPLC (H20 / CH3CN, see conditions below) and the pure product 18 was isolated as a yellow solid (61 mg, 26% yield). MHz, CDCI3) d 8.76 (s, 1H), 7.88 (d, J = 3.9 Hz, 1H), 7.78 (d, J = 4.0 Hz, 1H), 7. 75 (d, J = 8.7 Hz, 2H), 7.51 - 7.48 (m, 2H), 7.43 (s, 1H), 7.33 (d, J = 8.6 Hz, 2H), 7.20 (s, 1H), 7.15 (s, 1H), 6.86 (s, 1H), 6.84 (s, 1H), 6.74 (s, 1H), 6.68 (s, 2H), 6. 62 (s, 1H), 4.69 (p, J = 7.1 Hz, 1H), 4.41 - 4.24 (m, 5H), 4.24 - 4.16 (m, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.83 (t, J = 7.2 Hz, 4H), 3.67 - 3.56 (m, 33H), 3.55 3.49 (m, 1H), 3.39 (dt, J = 14.0, 7.0 Hz, 1H), 3.10 (dd, J = 15.0, 11. 6 Hz, 1H), 2.89 (dd, J = 16.9, 3.6 Hz, 1H), 2.75 - 2.64 (m, 1H), 2.51 (t, J = 7.2 Hz, 2H), 2.48 - 2.44 (m, 1H), 2.44 - 2.38 (m, 1H), 2.28 (dt, J = 13.3, 6.8 Hz, 1H), 1.47 (s, 1H), 1.46 (d, J = 7.1 Hz, 3H), 1.02 (dd, J = 10.7, 6.9 Hz, 6H), 0.82 - 0.72 (m, 2H), 0.55 (q, J = 5.2 Hz, 2H). ES + = 1.39 min, m / z 1404.45 [M + H] +.
Example 8: Activity of released compounds Test of K562 Human K562 chronic myeloid leukemia cells were maintained in RPM1 1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine at 37 ° C in a humidified atmosphere containing 5% CO2 and incubated with a specified dose of drug for 1 hour. hour or 96 hours at 37 ° C in the dark. The incubation was terminated by centrifugation (5 min, 300 g) and the cells were washed once with drug-free medium. After treatment with the appropriate drug, the cells were transferred to 96-well microtiter plates (104 cells per well, 8 wells per sample). Then, the plates were kept in the dark at 37 ° C in a humidified atmosphere containing 5% of C02 The assay is based on the ability of viable cells to reduce a yellow soluble tetrazolium salt, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H- bromide. tetrazolium (MTT, Aldrich-Sigma), to a precipitate of insoluble purple formazan. After incubation of the plates for 4 days (to allow the control cells to increase in number approximately 10 times), 20 ml of MTT solution (5 mg / ml in phosphate buffered saline) was added to each well and the plates were further incubated for 5 h. The plates were then centrifuged for 5 min at 300 g and volume of the cell pellet medium was pipetted leaving 10-20 ml per well. DMSO (200 μm) was added to each well and the samples were shaken to ensure complete mixing. Next, the optical density was read at a wavelength of 550 nm in a Titertek Multiscan ELISA plate reader, and a dose response curve was constructed. For each curve, an IC 50 value was read as the dose required to reduce the final optical density to 50% of the control value.
The RelC compound has an IC 50 of less than 0.1 pM in this assay.
Example 9: Formation of conjugates General procedure of antibody conjugation Antibodies are diluted at 1-5 mg / ml in a reduction buffer (examples: phosphate buffered saline PBS, histidine buffer, sodium borate buffer, TRIS tarapón). A freshly prepared solution of TCEP (tris (2-carboxyethyl) phosphine hydrochloride) is added to selectively reduce the cysteine disulfide bridges. The amount of TCEP is proportional to the target level of reduction, within 1 to 4 molar equivalents per antibody, generating 2 to 8 reactive thiols. After the reduction for several hours at 37 ° C, the mixture is cooled to room temperature and the excess drug-linker (A, B, C) is added as a diluted DMSO solution (final DMSO content up to 10 ° C). % in volume / volume of reaction mixture). The mixture is stirred gently at either 4 ° C or at room temperature for the appropriate time, usually 1-3 hours. Excess reactive thiols can be reacted with a 'thiol cap reagent' such as N-ethylmaleimide (NEM) at the end of the conjugation. The antibody-drug conjugates are concentrated using centrifuged concentration filters with a molecular weight cutoff of 10 kDa or higher, then purified by tangential flow filtration (TFF) or Rapid Liquid Protein Chromatography (FPLC). The corresponding antibody-drug conjugates can be determined by High Efficiency Liquid Chromatography (HPLC) or Ultra High Efficiency Liquid Chromatography (UHPLC) analysis to evaluate the ratio of drug to antibody (DAR). using reverse phase chromatography (RP) or Hydrophobic Interaction Chromatography (HIC), coupled to UV-Visible, Fluorescence or Mass Spectrometer detection; the level of aggregates and the purity of the monomers can be analyzed by HPLC or UHPLC using size exclusion chromatography coupled with UV-Visible detection, Fluorescence or Mass Spectrometer. The final concentration of the conjugates is determined by a combination of spectroscopic (absorbance at 280, 214 and 330 nm) and biochemical (BCA bicinchoninic acid assay, Smith, PK, et al (1985) Anal. Biochem. 150 (1) : 76-85, using an IgG antibody of known concentration as a reference). The antibody-drug conjugates are generally sterilized by filtration using 0.2 mm filters under aseptic conditions, and stored at + 4 ° C, -20 ° C or -80 ° C.
Examples of particular conjugations are described below.
ADC1A Antibody 1 (15 mg, 100 nanomoles) is diluted in 13.5 ml of a reduction buffer containing 10 mM sodium borate at pH 8.4, 2.5 mM EDTA and a final antibody concentration of 1.1 mg / ml. A 20 mM solution of TCEP is added (2 molar equivalents / antibody, 200 nanomoles, 10 ml) and the reduction mixture is heated at 37 ° C for one hour in an orbital incubator. After cooling to room temperature, A is added as a solution of DMSO (5 molar equivalents / antibody, 510 nanomoles, in 1.2 ml of DMSO). The solution is mixed 3 hours at room temperature, and then quenched by the addition of N-ethylmaleimide (NEM, 10 molar equivalents, 1000 nanomoles, 100 ml at 10 mM), then transferred to a 50 kDa centrifugal filter. of MWCO Amicon Ultracell of 15 i, is concentrated at approx. 2.0 ml and injected into an AKTA ™ FPLC using a GE Healthcare XK16 / 70 column filled with Superdex 200 PG, eluting with 1.5 ml / min of phosphate buffered saline (PBS) sterilized by filtration. The fractions corresponding to the ADC1A monomer peak are pooled, analyzed and sterilized by filtration. The BCA assay gives a final ADC1A concentration at 1.25 mg / ml in 10.0 ml, and the mass obtained is 12.5 mg (83% yield). Analysis of UHPLC in a Shimadzu Prominence system using an Agilent PLRP-S 1000 A 8 μm 150 x 2.1 mm column eluting with a gradient of water and acetonitrile over a reduced sample of ADC1A at 280 nm and 330 nm (drug specific) linker) shows a mixture of light and heavy chains linked to several molecules of A, consistent with a drug-to-drug ratio antibody (DAR) of 2.5 molecules of A per antibody. SEC analysis on an AKTA ™ FPLC using a GE Healthcare XK16 / 70 column filled with Superdex 200 PG, eluting with sterile phosphate buffered saline (PBS) by filtration on a sample of ADC1A at 280 nm shows a purity of monomer of 99.4% with 0.6% of aggregates.
ADC1B Antibody 1 (15 g, 100 nanomoles) is diluted in 13.5 ml of a reduction buffer containing 10 mM sodium borate at pH 8.4, 2.5 mM EDTA and a final antibody concentration of 1.1 mg / ml. A 10 mM solution of TCEP (3 molar equivalents / antibody, 300 nanomoles, 30 ml) is added and the reduction mixture is heated at 37 ° C for two hours in an orbital incubator. After cooling to room temperature, B is added as a solution of DMSO (7 molar equivalents / antibody, 700 nanomoles, in 1.0 ml of DMSO). The solution is mixed 3 hours at room temperature, and then transferred to a 50 kDa centrifuge filter of MWCO Amicon Ultracell of 15 ml, concentrated to approx. 2.0 ml and injected into an AKTA ™ FPLC using an XK16 column. 70 of GE Healthcare filled with Superdex 200 PG, eluting with 1.5 ml / min of phosphate buffered saline (PBS) sterilized by filtration. The fractions corresponding to the ADC1B monomer peak are pooled, analyzed and sterilized by filtration. The BCA assay gives a final ADC1B concentration at 1.57 mg / ml in 6.3 ml, and the mass obtained is 9.9 mg (66% yield). Analysis of UHPLC in a Shimadzu Prominence system using an Agilent PLRP-S 1000 A 8 um 150 x 2.1 m column that elutes with a gradient of water and acetonitrile over a reduced sample of ADC1B at 280 nm and 330 nm (drug specific) linker) shows a mixture of light and heavy chains linked to several B molecules, consistent with a drug to antibody ratio (DAR) of 2.8 B molecules per antibody. SEC analysis on an ARTA ™ FPLC using a GE Healthcare XK16 / 70 column filled with Superdex 200 PG, eluting with sterile phosphate buffered saline (PBS) by filtration on a sample of ADC1B at 280 nm shows a purity of monomer of 96.6% with 3.4% of aggregates.
ADC1C Antibody 1 (1.0 mg, 6.7 nanomoles) is diluted in 0.9 ml of a reduction buffer containing 10 mM sodium borate at pH 8.4, 2.5 m EDTA and a final antibody concentration of 1.1 mg / ml. A 1 mM solution of TCEP (3 molar equivalents / antibody, 300 nanomoles, 30 ml) is added and the reduction mixture is heated at 37 ° C for 1.5 hours in an orbital incubator. After cool to room temperature, C is added as a solution of DMSO (10 molar equivalents / antibody, 67 nanomoles, in 0.1 i of DMSO). The solution is mixed for 3 hours at room temperature, and then quenched by the addition of N-ethylmaleimide (NEM, 37 molar equivalents, 250 nanomoles, 10 ml to 25 mM), then injected into an AKTA ™ FPLC using a GE Healthcare XK16 / 70 column filled with Superdex 200 PG, eluting with 1.5 ml / min of phosphate buffered saline (PBS) sterilized by filtration. The fractions corresponding to the ADClC monomer peak are pooled, transferred to a 50 kDa centrifugation filter of MWCO Amicon Ultracell of 15 ml, concentrated to ca. 1.0 ml, are analyzed and sterilized by filtration. The BCA assay gives a final ADClC concentration at 0.63 mg / ml in 1.0 ml, and the mass obtained is 0.63 mg (63% yield). The UHPLC analysis on a Shimadzu Prominence system using an Agilent PLRP-S 1000 A 8 or 150 x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ADClC at 280 nm and 330 nm (drug specific) linker) shows a mixture of light and heavy chains linked to several C molecules, consistent with a drug to antibody ratio (DAR) of 2.9 C molecules per antibody. SEC analysis in an AKTA ™ FPLC using a column GE Healthcare XK16 / 70 filled with Superdex 200 PG, eluting with sterile phosphate buffered saline (PBS) by filtration on a sample of ADC1C at 280 nm shows a monomer purity of 99.0% with 1.0% aggregates.
ADC2A Antibody 2 (15 mg, 100 nanomoles) is diluted in 13.5 ml of a reduction buffer containing 10 mM sodium borate at pH 8.4, 2.5 mM EDTA and a final antibody concentration of 1.1 mg / ml. A 40 mM solution of TCEP (3 molar equivalents / antibody, 300 nanomoles, 7.5 ml) is added and the reduction mixture is heated at 37 ° C for one hour in an orbital incubator. After cooling to room temperature, A is added as a solution of DMSO (7 molar equivalents / antibody, 700 nanomoles, in 0.1 ml of DMSO). The solution is mixed for 2.5 hours at room temperature, then quenched by the addition of N-ethylmaleimide (NEM, 30 molar equivalents, 3000 nanomoles, 100 ml to 30 mM), then transferred to a 50-centimeter centrifuge filter. kDa of MWCO A icon Ultracell of 15 ml, concentrates at approx. 2.0 ml, and injected into an AKTA ™ FPLC using a GE Healthcare XK16 / 70 column filled with Superdex 200 PG, eluting with 1.5 ml / min of phosphate buffered saline (PBS) sterilized by filtration.
The fractions corresponding to the ADC2A monomer peak are pooled, concentrated using a 50 kDa centrifuge filter of MWCO A icon Ultracell of 15 ml, analyzed and sterilized by filtration. The BCA assay gives a final ADC2A concentration at 3.94 mg / ml in 2.7 ml, and the mass obtained is 10.6 g (71% yield). Analysis of UHPLC in a Shimadzu Prominence system using an Agilent PLRP-S 1000 A 8 μm 150 x 2.1 mm column eluting with a gradient of water and acetonitrile over a reduced sample of ADC2A at 280 nm and 330 nm (drug-specific) linker) shows a mixture of light and heavy chains linked to several molecules of A, consistent with a drug to antibody ratio (DAR) of 2.4 molecules of A per antibody. Analysis of UHPLC in a Shimadzu Prominence system using a UPLC BEH200 SEC Waters Acquity 1.7 um 4.6 x 150 mm column eluting with sterile phosphate buffered saline (PBS) by filtration on a sample of ADC2A at 280 nm shows a monomer purity of the 97.5% with 1.9% aggregates.
As used herein, "Antibody 1" is an anti-Her2 antibody that comprises a VH domain having the sequence in accordance with SEQ ID NO. 1 and a VL domain having the sequence in accordance with SEQ ID NO.2.
As used in this document, "Antibody 2" is an anti-CD25 antibody ("Simulect") comprising a VH domain having the sequence according to SEQ ID NO.3 and a VL domain having the sequence according to SEQ ID NO.4.
Example 10: Efficacy studies of ADC in vitro The cytotoxicity of ADC2A was evaluated in a cytotoxicity assay as described above, and the results are shown in Figure 1. or represents the cell line expressing the antigen (SU-DHL-1 cells of the German Collection of Microorganisms and Cell Cultures DSMZ of the Leibniz Institute), and? represents the cell line that does not express the antigen (Daudi cells of the American Type Culture Collection), and the error bars indicate ± standard deviation.
Example 11: Efficacy studies of ADC in vivo CB.17 SCID mice, aged 8-12 weeks, are injected subcutaneously with 1 mm3 tumor fragments derived from the BT-474 cell line on the flank. When tumors reach an average size of 100-150 mg, treatment is started. The mice are weighed twice a week. The tumor size is measured twice a week. The animals are monitored individually. The final point of the experiment is a tumor volume of 1000 mm3 or 60 days, whichever appears first. Those who respond can be followed more time.
Groups of 10 xenografted mice are injected i.v. with 0.2 ml of antibody-drug conjugate (ADC), or naked antibody, in phosphate-buffered saline (vehicle) or with 0.2 ml of vehicle alone. The concentration of ADC is adjusted to give, for example, 0.3 or 1.0 mg of ADC / kg body weight in a single dose. Three identical doses can be administered to each mouse at intervals of, for example, 1 week.
Figure 2 shows the effect on mean tumor volume in groups of 10 mice dosed with ADC1A at 0.3 (yellow) or 1.0 mg / kg (purple) compared to vehicle controls (black) or naked antibody (blue).
Figure 3 shows the effect on mean tumor volume in groups of 10 mice dosed with ADC1B at 0.3 (gray) or 1.0 mg / kg (purple) compared to vehicle controls (black) or naked Ig (blue).
All the documents and other references mentioned above are incorporated herein by way of reference.
Abbreviations Ac acetyl Acm acetamidomethyl Alloc allyloxycarbonyl Boc di-tert-butyl dicarbonate tere-butyl Bzl benzyl, where Bzl-OMe is methoxybenzyl and Bzl-Me is methylbenzene Cbz or Z benzyloxycarbonyl, wherein Z-Cl and Z-Br are chloro- and bromobenzyloxycarbonyl, respectively DMF N, N-dimethylformamide Dinitrophenyl Dnp DTT dithiothreitol Fmoc 9H-fluoren-9-ylmethoxycarbonyl Imp imine protective group W-10: 3- (2-methoxyethoxy) propanoate-Val-Ala-PAB MC-OSu maleimidocaproyl-O-A / -succinimide Moc methoxycarbonyl MP maleimidopropanamide Mtr 4-methoxy-2,3,6-trimethylbenzenesulfonyl PAB para-aminobenzyloxycarbonyl PEG ethyleneoxy PNZ carbamate of p-nitrobenzyl Psec 2- (phenylsulfonyl) ethoxycarbonyl TBDMS tert-butyldi ethylsilyl TBDPS tert-butyldiphenylsilyl Teoc 2- (trimethylsilyl) ethoxycarbonyl Tosyl cough 2,2,4-trichloroethoxycarbonyl chloride Trityl Trt Xanthin Xanthyl

Claims (18)

1. A compound, which is selected from A: TO B: and C: and salts and solvates thereof.
2. A conjugate of formula ConjA: ConjC wherein CBA represents a cell binding agent.
3. The conjugate according to claim 2, characterized in that the cell binding agent is an antibody or an active fragment thereof.
4. The conjugate according to claim 3, characterized in that the antibody or antibody fragment is an antibody or antibody fragment for a tumor-associated antigen.
5. The conjugate according to claim 3, characterized in that the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from (1) - (88): (1) BMPR1B; (2) El6; (3) STEAP1; (4) 0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; (8) PSCA hlg; (9) ETBR; (10) MSG783; (11) STEAP2; (12) TrpM4; (13) CRYPT; (14) CD21; (15) CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; (20) IL20R-alpha; (21) Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29) CXCR5; (30) HLA-DOB; (31) P2X5; (32) CD72; (33) LY64; (34) FcRHl; (35) IRTA2; (36) TENB2; (37) PSMA - FOLH1 (38) SST; (38.1) SSTR2; (38.2) SSTR5; (38.3) SSTR1; (38.4) SSTR3; (38.5) SSTR4; (39) ITGAV; (40) ITGB6; (41) CEACAM5; (42) MET; (43) MUC1; (44) CA9; (45) EGFRvIII; (46) CD33; (47) CD19; (48) IL2RA; (49) AXL; (50) CD30-TNFRSF8; (51) BCMA-TNFRSF17; (52) CT Ags - CTA; (53) CD174 (Lewis Y) -FUT3; (54) CLEC14A; (55) GRP78-HSPA5; (56) CD70; (57) Stem cell-specific antigens; (58) ASG-5; (59) ENPP3; (60) PRR4; (61) GCC-GUCY2C; (62) Liv-1 - SLC39A6; (63) 5T4; (64) CD56-NCMA1; (65) CanAg; (66) F0LR1; (67) GPNMB; (68) TIM-1 - HAVCR1; (69) RG-l / Mindina prostate tumor target Mindina / RG-1; (70) B7-H4 - VTCN1; (71) PTK7; (72) CD37; (73) CD138-SDC1; (74) CD74; (75) Claudinas - CLs; (76) EGFR; (77) Her3; (78) RON - MST1R; (79) EPHA2; (80) CD20-MS4A1; (81) Tenascin C-TNC; (82) FAP; (83) DKK-1; (84) CD52; (85) CS1 - SLAMF7; (86) Endoglina- ENG; (87) Annexin Al - ANXA1; (88) V-CAM (CD106) - VCAM1.
6. The conjugate according to any of claims 2 to 5, characterized in that the antibody or antibody fragment is an antibody manipulated with cistern.
7. The conjugate according to any of claims 2 to 6, characterized in that the drug (p) loading of drugs (D) to an antibody (Ab) is an integer from 1 to about 8.
8. The conjugate according to claim 7, characterized in that p is 1, 2, 3, or 4.
9. A composition comprising a mixture of drug conjugate compounds according to any of claims 2 to 8, characterized in that the average drug load per antibody in the mixture of antibody-drug conjugate compounds is from about 1 to about 8. .
10. The conjugate according to any of claims 2 to 8 or the conforming composition with claim 9, for use in therapy.
11. The conjugate according to any of claims 2 to 8 or the composition according to claim 9, for use in the treatment of a Proliferative disease in a subject.
12. The conjugate or composition according to claim 11, characterized in that the disease is cancer.
13. A pharmaceutical composition characterized in that it comprises the conjugate according to any of claims 2 to 8 or the composition according to claim 9, and a pharmaceutically acceptable diluent, carrier or excipient.
14. The pharmaceutical composition according to claim 13, characterized in that it further comprises a therapeutically effective amount of a chemotherapeutic agent.
15. Use of the conjugate according to any of claims 2 to 8 or the composition according to claim 9 in the preparation of a medicament for use in the treatment of a proliferative disease in a subject.
16. A method for treating cancer comprising administering to a patient the pharmaceutical composition according to claim 14.
17. The method according to claim 16, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.
18. A method for preparing a conjugate of according to any of claims 2 to 8, the method characterized in that it comprises the step of reacting a cell binding agent with the compound A, B or C as defined in claim 1.
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